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). In practice, this means that preparation, analysis, and detection can all take place within a single droplet. Compared with bulk experiments, reactions are faster, less reagent is wasted, and many tests can be run at the same time. The method is also more sensitive and gives researchers tighter control at each step [1, 2].
Droplet microfluidics advantages
Traditional life science methods are often costly and depend on complex cell labeling. Sample preparation can take time, which may compromise cell health and increase the risk of contamination. In addition, these approaches are limited in their detection ability [3]. On the other side, miniaturized plates can only support static cell cultures, unable 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 are straightforward and easy to set up, which makes them suitable for creating neat arrays of single cells. However, it can be difficult to deliver liquids on the spot because the reagents have to be well spread across the whole array. 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 widely used for separating cell types, but it mostly works on bulk populations. That makes it hard to study individual cells. It also relies on fluorescent markers, which often demand preparation that can be harsh on the cells [5].
With droplet microfluidics, researchers can study both what’s happening inside the cell (Figure 3) and also at the molecules it releases, without the need for aggressive 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].
Rivello and colleagues analyzed the metabolism of individual circulating stromal cells from prostate cancer patients using droplet microfluidics. By measuring pH changes and sequencing mRNA at the single-cell level, they were able to detect highly active cells, highlighting 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 [12].
DNA construction in droplets
Microarrays were considered a great advancement in reducing costs and minimizing reaction volumes; however, microfluidics introduced 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 [14]. Yehezkel and colleagues were able to build double-stranded DNA from 160-base-pair pieces inside nanoliter droplets [15].
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 [16].
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 and colleagues were able to detect SARS-CoV-2 with high sensitivity and accuracy using a chip that automates nucleic acid extraction and digital droplet PCR [17].
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 [19].
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 [20] (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 [21]. 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 the single-cell level (Figure 8). This approach helps identify rare immune cells and select the best candidates for cell-based therapies.
One innovative technique consists of anchoring cholesterol-linked antibodies to the cell surface before encapsulation. Cytokines secreted by immune cells 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 [22].
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 [23].
Enzyme Kinetics
Recent studies have highlighted the use of droplet microfluidics for enzyme-directed evolution, the study of novel catalytic activity, and the enhancement of protease and peptidase properties (Figure 9).
Okal and colleagues used a high-throughput droplet-based platform to screen libraries of Angiotensin-converting enzyme 2 (ACE2) variants directly on their peptide. The authors were able to highlight beneficial mutations, such as K187T, that significantly enhanced ACE2’s catalytic activity. Their approach demonstrates how microfluidics can facilitate the precise and scalable development of therapeutic enzymes [25].
Schnettler and co-workers recently showed an advancement in the use of droplet microfluidics for enzyme evolution. They took a metal-free α/β-hydrolase and, through droplet screening, turned it into a phosphotriesterase. The reaction they achieved was about a billion times faster than the uncatalyzed version, which made it possible to quickly identify the most active variants [26].
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 requires 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 daily 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 can not handle.
- Chip material limitations: Choosing the right material can be tricky. Glass chips offer good surface modification options but are expensive (~$500/chip). On the other hand, 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: Droplet microfluidics is mostly based on Newtonian fluids, which don’t translate well to real biological samples like blood.
- Interfacial tension: Interfacial tension is often considered as constant, which isn’t valid for dynamic or temperature-varying systems (e.g., PCR).
- Screening and sorting: Among droplet manipulations, screening and sorting are the most challenging, as only a small fraction of droplets among millions encapsulate the desired cells [28].
Conclusion - What’s next for droplet microfluidics?
Droplet microfluidics has enabled 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
