Creating a Large Continuous Droplet Using Screen
Droplet barcoding: tracking mobile micro-reactors for high-throughput biology
Droplet microfluidics has become a powerful analytical platform in biological research for conducting high-throughput screening in millions of discrete micro-reactors per hour. While the method facilitates faster and cheaper testing than conventional microtiter plates, the mobile nature of droplets makes micro-reaction tracking a notable challenge. To address this, researchers are developing a variety of tracking methods, ranging from organizing droplets into an index or labeling droplets with a barcode. The optimal tracking approach depends on the criteria for each specific application. Considerations include the requisite assay readout, throughput, droplet library size, reagent tracking and more. In this review, we summarize different strategies for droplet micro-reaction tracking and comment on promising future approaches in droplet barcoding. Topics range from indexing droplets by sequence or in an array, labeling droplets with barcodes, and reagent barcoding to track the input conditions in parametric screens.
Introduction
In the midst of an influenza epidemic and laboratory supply shortage, Dr Gyula Takátsy introduced microtiter plates in 1950 [1,2]. Since then, microtiter plates have become a keystone analytical platform in research and medicine due to their standardized, sterile and easy-to-interface array of discrete reaction chambers. In the last several decades, biological research has advanced toward precision science, with researchers directly interrogating the innumerable cellular and molecular interactions that precipitate life. The analytical throughput demands of many biochemical applications, ranging from drug discovery to studying cellular heterogeneity, now far exceed the capacity of microtiter plates that host volumes in the microliter range. Furthermore, scaling down from microliter multiwell plates to nanoliter microarrays introduces severe limitations with respect to pipetting or spotting as well as potential evaporation of fluids. Droplet microfluidics has emerged as an alternative approach that exhibits many of the strengths of microtiter plates with several orders of magnitude improvements in throughput and cost.
Using simple microfluidic architectures and flow controls, microfluidic approaches can generate millions of picoliter mono-disperse water-in-oil droplets per hour. Each droplet is stabilized by water–oil interfacial surfactants or nanoparticles [3•] to ensure that the micro-reactions remain discrete. The low-volume and reagent consumption per test can reduce screening costs by a million-fold [4]. A growing portfolio of reagent delivery and droplet generation approaches has enabled precise control of droplet reagent composition, which is necessary for complex assays [5]. Moreover, significant progress has been made on adapting important readout modalities to droplet microfluidics, including label-free approaches such as mass spectrometry [6,7•], absorption [8] and Raman spectroscopy [9]. Combination with sorting modules has led to a powerful microfluidic cytometry equivalent referred to as fluorescence-activated droplet sorting [10•].
While droplet microfluidics has managed to fulfill many of the critical features of microtiter plates, it generates new challenges for reaction tracking. Mobile droplet micro-reactors lack the well-controlled spatial index of well plates. For functional genetic screens, it is essential to link an observed phenotype in a droplet with the underlying molecular sequence. Many enzymatic or cell growth assays require time-course measurements per individual reactions, which is impossible to achieve without the use of a droplet index. Parametric or combinatorial screens where reaction input conditions are systematically varied also require a robust approach for tracking the reagent composition of each reaction.
For certain assays, a droplet barcode is required, where a code refers to a specific droplet or its contents. In others, a droplet index is sufficient, where an organization method makes droplets uniquely identifiable. These terms and others used in the review are defined in Box 1. To avoid additional readouts, tracking schemes are optimally implemented such that the input conditions of the droplet, the barcode or index, and assay performance can be readout simultaneously. Furthermore, as droplet microfluidic readouts vary widely for different applications — from fluorescence microscopy to mass spectrometry — there are different requirements for appropriate tracking methods. In this review, we discuss both the utility and limitations of various droplet micro-reaction tracking methods and comment on potential future developments in droplet barcoding (Figure 1).
Section snippets
Droplet sequence
Tracking droplets in sequence is the simplest way to index droplets in a microfluidic channel but it is also susceptible to disruption since droplet integrity and sequence must be maintained throughout the platform. Off-chip droplet collection that disrupts sequence or microfluidic designs that can result in droplet splitting are incompatible. In one example of droplet sequence indexing, combinatorial droplets for enzyme screening were deterministically generated at several hertz using an array
Droplet array
As an alternative to indexing by flow sequence, droplets can also be indexed on a surface. Arraying droplets within microwells [14••], chemical micropatterns [7•], microdevice designs [15,16], dielectric trapping [17••], or many other approaches [18] allows for a spatial index for each micro-reaction, similar to that of microtiter plates. This approach adds another process step for deposition and can limit the throughput to thousands of droplets per hour. Nonetheless, this method provides
Particle barcode
A conceptually simple strategy for micro-reactor tracking is the use of a particle barcode to track the contents of each droplet. This is analogous to stores using stickers with the Universal Product Code – encoded with sequential vertical lines – to label and track inventory [22]. A particle barcoded droplet index requires the following: a coding strategy compatible with the assay readout; a sufficiently sized particle with chemical properties that are compatible with the micro-reaction; and a
Reagent barcode
Droplet microfluidics can be used for optimizing reactions over a large parameter space of conditions, which is useful in applications like drug screening, toxicity testing, and chemical synthesis. During such a screen, tracking the input conditions of each droplet with robust internal concentration standards is more important than tracking the individual droplets. When a well-calibrated set of internal standards is used for each reagent, relatively simple non-deterministic approaches can
Conclusions
There are many demonstrated and emerging new technologies to track droplets. However, many of these approaches are not routinely used today. Limitations include the number of barcodes that can be encoded and produced, the time and complexity required to incorporate a barcode, and the requirement that the barcode should not interfere with the actual assay readout from the micro-reactor. For example, a fluorescence assay reduces the wavelength-space of optical barcodes or the barcode itself could
Conflict of interest statement
Nothing declared.
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
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• of special interest
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•• of outstanding interest
Acknowledgement
We gratefully acknowledge funding from the Swiss National Science Foundation (NCCR Molecular Systems Engineering).
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