Digital Assays Part II: Digital Protein and Cell Assays

Abstract

A digital assay is one in which the sample is partitioned into many containers such that each partition contains a discrete number of biological entities (0, 1, 2, 3, . . .). A powerful technique in the biologist's toolkit, digital assays bring a new level of precision in quantifying nucleic acids, measuring proteins and their enzymatic activity, and probing single-cell genotype and phenotype. Where part I of this review focused on the fundamentals of partitioning and digital PCR, part II turns its attention to digital protein and cell assays. Digital enzyme assays measure the kinetics of single proteins with enzymatic activity. Digital enzyme-linked immunoassays (ELISAs) quantify antigenic proteins with 2 to 3 log lower detection limit than conventional ELISA, making them well suited for low-abundance biomarkers. Digital cell assays probe single-cell genotype and phenotype, including gene expression, intracellular and surface proteins, metabolic activity, cytotoxicity, and transcriptomes (scRNA-seq). These methods exploit partitioning to 1) isolate single cells or proteins, 2) detect their activity via enzymatic amplification, and 3) tag them individually by coencapsulating them with molecular barcodes. When scaled, digital assays reveal stochastic differences between proteins or cells within a population, a key to understanding biological heterogeneity. This review is intended to give a broad perspective to scientists interested in adopting digital assays into their workflows.

Formation of uniform reaction volumes using concentric amphiphilic microparticles

“…a microbead and a target cell or molecule inside a droplet approaches ~1% of the entire population of droplets, whereas the remaining droplets would have undesired combinations of the components. [4,14,15] Moreover, for multiplexing, multiple types of microbeads with distinct barcoding signatures should be encapsulated in separate droplets with the targets of interest, which is further limited by multiplicative probabilities to triple or larger Poisson distributions for duplex or greater multiplexing. Therefore, an instrument-free compartmentalization system that forms uniform droplets embedded with a single solid phase per droplet can address many of the challenges with current approaches.…”

Massively parallel encapsulation of single cells with structured microparticles and secretion-based flow sorting

“…It was noted that this seeding approach led to cell occupancies that closely followed Poisson statistics ( Figure 2c), as is expected for loading of single cells into microfluidically-generated droplets. 30,31 Therefore, by controlling the cell seeding density, we can control occupancy such that most particles have either 0 or 1 cells associated with them.…”

Multi-dimensional digital bioassay platform based on an air-sealed femtoliter reactor array device

“…Digital bioassay is one such method widely used in recent years (2)(3)(4)(5)(6). In this method, analytes (e.g., nucleic acids, enzymes, or other proteins) are diluted and partitioned into a large number of small compartments, which results in "0," compartments containing no analytes, or "1," compartments containing only a single analyte.…”

“…Upon amplifying and detecting signals from these "digitalized" compartments, we can quantify analytes in a single-molecule resolution simply by counting the "1" compartments. Digital bioassay has enabled ultrasensitive detection of diverse analytes, including nucleic acids (called digital PCR [5,[7][8][9][10][11]), proteins (called digital ELISA [6,12,13]), and even influenza virus particles (14,15). The most commonly used compartment in digital bioassay is a droplet emulsion (9,10,16,17), typically a water droplet separated by a continuous oil phase.…”