Droplet microfluidics in action
From innovative applications to core challenges
Writer
Celeste Chidiac, PhD
Keywords
Microfluidic Devices, Intelligent Microfluidics, Artificial Intelligence, Machine Learning
Author
Celeste Chidiac, PhD
Publication Date
June 26, 2025
Keywords
Intelligent Microfluidics
Deep Learning
Microfluidic Devices
Artificial Intelligence
Machine Learning
Droplet Microfluidics
Single-cell analysis
Drug screening
DNA construction
Tissue engineering
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Introduction to droplet microfluidics
Droplet microfluidics is a technology for studying the generation, manipulation, and application of femtoliter to nanoliter drops encapsulated into monodisperse droplets (Figure 1). It combines sample preparation, analysis, and detection in a single, compact system. Unlike traditional bulk methods, it accelerates reactions, reduces the amount of chemicals required, and facilitates the simultaneous execution of multiple tests. It also improves sensitivity and allows for greater control over each step of the process [1].
Droplet microfluidics advantages
Traditional life science methods often depend on expensive machines and complex cell labeling techniques. Preparing samples for analysis can be a time-consuming process, which may compromise cell health and increase the risk of contamination. These approaches are not only costly and technically demanding but also limited in their ability to effectively detect what is being studied [3]. In addition, miniaturized microtiter plates, while useful, can only support static cell cultures, restricting their ability to replicate the dynamic extracellular microenvironment necessary for accurate biological studies.
Microfluidics is quickly becoming a go-to tool in life sciences for good reason. It uses small amounts of material, delivers highly sensitive results, and fits easily into larger lab systems. It can be used to perform 3D cell culturing through continuous infusion, simulating the microenvironment associated with cell physiology in vivo [4].
Microwell-based methods, by contrast, are known for being straightforward and easy to set up. They’re great for creating neat arrays of single cells. But there’s a catch: the reagents have to be spread across the whole array, which makes it hard to deliver liquids on the spot. That’s a problem if you’re trying to track fast changes in cell behavior.
That’s where droplet microfluidics shines. It’s fast, reliable, and doesn’t waste precious reagents. More importantly, it provides researchers with tight control over experiments and is especially useful when studying how cells react in real-time, making it a smart upgrade for many types of cell research [4] (Figure 2).
Droplet microfluidics applications
Cell sorting & single-cell analysis
Fluorescence-activated cell sorting (FACS) is a widely used technique for separating different types of cells. But while it’s effective, it has some serious limitations. FACS usually works on bulk populations, which makes it hard to track the behavior of individual cells. It also relies on fluorescent markers that often require harsh preparation that can damage or even kill the cells [5].
Droplet microfluidics offers a promising alternative. By generating tiny, customizable droplets, researchers can study both what is happening inside a single cell (Figure 3) and what it is releasing into its surroundings, all without exposing cells to harmful treatments [1].
Recently, scientists have begun combining droplet microfluidics with traditional FACS, creating hybrid systems that blend FACS’s speed with the precision of single-cell droplet analysis. The result: much higher resolution and control.
For example, Lan and colleagues used droplet microfluidics combined with permeable microgels to sequence the genomes of 50,000 single cells. Their setup enabled them to gently break open the cells and isolate their DNA while preserving its integrity [6].
In another study, Rivello et al. applied droplet microfluidics to analyze the metabolism of individual circulating stromal cells from prostate cancer patients. By measuring pH changes and sequencing mRNA at the single-cell level, they were able to pinpoint highly active cells, revealing the technique’s strong potential for cancer diagnostics [7].
Drug screening
In microfluidic single-cell drug screening, selected compounds are added to isolated individual cells, not to large cell populations, as is typically done in flow cytometry [8]. This approach eliminates cell-cell interactions and enables the isolation of drug-resistant cells, based on their variable drug sensitivity [4].
For example, Sarkar et al. used a droplet microfluidics setup to study how breast cancer cells absorb and react to doxorubicin. They found that sensitive cells absorbed more of the drug and were more likely to die, often showing bursts of uptake. In contrast, drug-resistant cells showed lower uptake and retention [9].
Building on this idea, Bithi et al. developed a simple, pipette-based method to isolate cells and create droplet arrays for analyzing tiny tumor samples (Figure 4). They found that while drug uptake varied among individual cells, cell death only happened once a critical intracellular concentration was reached [10].
Tissue engineering
By creating 3D structures like tumor spheroids, stem cell spheroids, and organoids (Figure 5), researchers can closely replicate cell behavior, interactions, and differentiation in real human tissues. When combined with cancer-associated fibroblasts (CAFs), these cells provide new insights into tumor resistance to drugs, and allow for the study of tumor stress behaviors under hypoxic conditions [11].
Pushing things even further, researchers are now turning to “organoid-on-a-chip” systems for greater control over nutrient flow and mechanical forces. A team led by Wang developed a one-step method to grow uniform liver organoids from human-induced pluripotent stem cells (hiPSCs) using double emulsion and microfluidic valves [13].
DNA construction in droplets
Microarrays represented a significant step forward in reducing costs by minimizing reaction volumes; however, microfluidics introduces a new level of precision, enabling the individual manipulation of reaction chambers (Figure 6). Digital microfluidic platforms can now automate entire DNA workflows, like constructing gene libraries or assembling large DNA fragments [15]. Yehezkel et al. even managed to build double-stranded DNA from 160-base-pair pieces inside nanoliter droplets [16].
Coelho and colleagues created a hybrid system that combines digital and droplet microfluidics. First, the digital part carefully mixes the reagents, and then those mixtures are sent into the droplet module, which produces stable, nanoliter-sized droplets. While traditional droplet devices can create a wider variety of droplet sizes and speeds, this hybrid setup provides more consistent and controlled droplets, without slowing down the process [17].
These systems aren’t just for assembling DNA. Shih and his team created a chip that could mix DNA and enzymes, incubate them in winding microchannels, and even deliver electrical pulses to insert the DNA into cells automatically. Meanwhile, Ma et al. developed a chip that automates nucleic acid extraction and digital droplet PCR, enabling the detection of viruses like SARS-CoV-2 with high sensitivity and accuracy [19].
CRISPR-powered microfluidics
Microfluidics is proving to be a game-changer for CRISPR-based gene editing, enabling researchers to automate and miniaturize crucial steps, such as RNA delivery, transfection, and clone selection. This integration leads to faster workflows, reduced reagent use, and more reproducible results – key advantages for scaling up genome engineering [21].
Iwai et al. developed a chip that combines gene editing and testing. It uses a grid of 100 electrodes to move droplets around and zap cells with electric pulses. When they tested it on E. coli bacteria, they were able to enhance metabolite production [22] (Figure 7).
Another team, led by Chen, combined CRISPR with magnetic particles and microfluidics to develop a small device that detects SARS-CoV-2 in under 30 minutes using just 100 µL of sample [23]. It showed high sensitivity, highlighting the potential of CRISPR-microfluidic platforms for rapid, point-of-care diagnostics.
Immunotherapy research
Droplet microfluidics enables highly sensitive functional assays at single-cell resolution (Figure 8). This approach enhances the ability to identify rare yet powerful immune cells, facilitating the selection of optimal candidates for cell-based therapies.
One innovative technique involves anchoring cholesterol-linked antibodies to the surface of each cell before encapsulation. As immune cells secrete cytokines, these molecules are captured directly on the same cell’s surface, eliminating signal mixing between neighboring cells and thereby helping to better distinguish between cell types and activation states [24].
In a similar effort, Yuan and colleagues used droplet microfluidics to co-culture natural killer (NK) cells with their target cells. This setup enabled them to precisely measure the release of interferon-gamma (IFN-γ), a key immune signaling molecule. Their method significantly reduced false results and enabled the sorting of activated cells using standard FACS [25].
Enzyme Kinetics
Recent studies have highlighted the benefits of using droplet microfluidics for enzyme-directed evolution, discovery of novel catalytic activity, and the enhancement of protease and peptidase properties (Figure 9).
In a recent study, Okal et al. used a high-throughput droplet-based platform to screen libraries of Angiotensin-converting enzyme 2 (ACE2) variants directly on their natural target, the Angiotensin-II peptide. This allowed them to pinpoint beneficial mutations, such as K187T, that significantly boosted ACE2’s catalytic activity. Their approach demonstrates how microfluidics can facilitate the precise and scalable development of therapeutic enzymes [27].
Schnettler et al. took enzyme evolution a step further, engineering a metal-free α/β-hydrolase into an efficient phosphotriesterase using microfluidic droplet screening. By applying ultrahigh-throughput selection, they achieved a 1 billion-fold rate improvement over the uncatalyzed reaction, enabling the rapid selection of highly active variants [28].
Challenges and future directions
Droplet microfluidics encounters some challenges that hinder its wider adoption (Figure 10).
- Application-specific requirements: Each application has very specific requirements for droplet functions, e.g., single-cell analysis needs tiny droplets, whereas tissue engineering needs much larger ones. Technologies for one application can’t simply be scaled or modified for another.
- Disconnection between developers and users: Engineers creating microfluidic systems may not fully understand the day-to-day needs of researchers, while users often find the systems too technical or rigid. Even a small change in an experiment might require a complete redesign of the chip—something most users aren’t equipped to handle.
- Chip material limitations: Glass chips offer good surface modification options but are expensive (~$500/chip). PDMS chips are cheap and optically transparent, but they are mechanically soft and prone to swelling in oil, which reduces accuracy and compatibility with certain chemicals.
- Wettability requirements: Droplet stability relies on surfactants and surface treatments, which need to be fine-tuned for each experiment.
- Fluid property dependency: Mostly based on Newtonian fluids; doesn’t translate well to real biological samples like blood.
- Interfacial tension: Often assumed constant, which isn’t valid for dynamic or temperature-varying systems (e.g., PCR).
- Screening and sorting: Among droplet manipulations, sorting is the most challenging, as it involves isolating rare target-containing droplets from millions. Since only a small fraction of droplets encapsulate desired cells, efficient screening and sorting are crucial [30].
Conclusion - What’s next for droplet microfluidics?
Droplet microfluidics has unlocked exciting new possibilities in biology, chemistry, and diagnostics. Its ability to precisely control tiny liquid amounts makes it ideal for high-throughput experiments, single-cell studies, and enzyme screening, saving time, reducing waste, and facilitating discoveries that would be hard to achieve with traditional methods. But alongside these strengths come real challenges. The technology can be complex to design and use, and experiments often need careful tuning to work properly. Even so, there are opportunities for smarter design, better collaboration, and creative problem-solving. As researchers continue to push the limits of what’s possible and make systems more reliable and user-friendly, droplet microfluidics is likely to become an even more powerful tool across science and medicine.
References
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Funding and Support
This review was written under the European Union’s Horizon research and innovation program under HORIZON-EIC-2023-PATHFINDEROPEN-01, grant agreement no. 101130747 (Bio-HhOST),
and ANR (Agence Nationale de la Recherche, project number ANR-23-CE10-0018-02) and the SNF (Schweizerischer Nationalfonds) in the framework of the AAPG2023 PRCI (Voxwrite).
This review was written by Celeste Chidiac, PhD.
Published in June 2025.
Contact: Partnership@microfluidic.fr
