Stationary nanoliter droplet array with a substrate of choice for single adherent/nonadherent cell incubation and analysis

Abstract

Microfluidic water-in-oil droplets that serve as separate, chemically isolated compartments can be applied for single-cell analysis; however, to investigate encapsulated cells effectively over prolonged time periods, an array of droplets must remain stationary on a versatile substrate for optimal cell compatibility. We present here a platform of unique geometry and substrate versatility that generates a stationary nanodroplet array by using wells branching off a main microfluidic channel. These droplets are confined by multiple sides of a nanowell and are in direct contact with a biocompatible substrate of choice. The device is operated by a unique and reversed loading procedure that eliminates the need for fine pressure control or external tubing. Fluorocarbon oil isolates the droplets and provides soluble oxygen for the cells. By using this approach, the metabolic activity of single adherent cells was monitored continuously over time, and the concentration of viable pathogens in blood-derived samples was determined directly by measuring the number of colony-formed droplets. The method is simple to operate, requires a few microliters of reagent volume, is portable, is reusable, and allows for cell retrieval. This technology may be particularly useful for multiplexed assays for which prolonged and simultaneous visual inspection of many isolated single adherent or nonadherent cells is required.single cell | nanoliter array | diagnostics C ommon single-cell analysis methods, such as flow cytometry and mass cytometry (1), offer high throughput and accurate single-cell marker quantification, yet they lack the ability to monitor large numbers of single cells continuously and simultaneously in performance-based assays (2, 3). Conventional microscopy may be used for these assays; however, in the case of single cells, they cannot analyze extracellular events, such as secretion. To achieve this, cells must be isolated in compartments that can sustain cell viability and growth while permitting conventional optical analysis over many hours to days. Dropletbased microfluidics, which enables single-cell encapsulation in nano-and subnanoliter droplets by surrounding microscopic aqueous medium with an immiscible carrier fluid (4-8), recently gained interest with the appearance of digital PCR (9-11). Much of the work thus far has been directed toward improving droplet manipulation capabilities (12-16). With these methods, droplets are mobile, and thus cytometry is performed under flow conditions (17), making continuous monitoring of single cells difficult. Continuous monitoring may be achieved by using stationary indexed droplets, but many current droplet immobilization techniques are limited by pressure coupling between droplet generation and capture events, as well as the requirement to adjust droplet volume to nanowell size (6,18,19). The vast majority of methods used to generate water-in-oil droplets begin by priming a continuous oil phase in a microfluidic channel followed by an injection of a dispersed (aqueous) medium (2...

A hydrogel beads based platform for single-cell phenotypic analysis and digital molecular detection

“…It was also noticed that, after 3 hours of incubation, the positive Gelbead fraction was 36.4±8.1%, which corresponds well with the theoretical fraction of Gelbeads (29%) encapsulating more than 1 cell. Based on the linear response of alamarBlue to the number of cells within the compartment (36), our results reasonably indicate that effective single cell phenotyping in Gelbeads is achievable within 4 hrs. However, 5 hr incubation lead to overly bright fluorescence and 92.9±2.7% bright Gelbeads, which was likely attributed to excessive incubation and the diffusion of metabolized fluorescent resorufin across the aqueous-oil interfacial barrier.…”

The Poisson distribution and beyond: methods for microfluidic droplet production and single cell encapsulation

“…197,198 Interestingly, encapsulation also allows cells to be used effectively as therapeutic agents in their own right, where encapsulated cells hold substantial promise for delivery of cell-produced drugs. 202 6 Summary and prospects A number of droplet production methods for the purpose of microfluidic encapsulation have been presented and discussed. These benefits are further enhanced in a core-shell capsule, where a hydrogel bead is encased in a secondary polymer shell to prevent ingress or egress of cells.…”

Reducing Uncertainty for Acute Febrile Illness in Resource-Limited Settings: The Current Diagnostic Landscape

“…59 Other bacterial culture techniques in early stages of development use hundreds of nanoliter-scale droplets that may dramatically reduce the size of a microbiology laboratory. 60,61 Instead of relying on variable visual inspection or a cumbersome microplate reader for enzyme-linked immunosorbent assay, technology has been developed to use smartphones to accurately read the results of serologic tests. 64,65 As with nucleic acid amplification test (NAAT), the ability to diagnose multiple infections allows for more cost-effective and streamlined diagnosis of acute febrile illness.…”