Microfluidic Droplets: From Basic Principles to Versatile Tools
Microfluidic droplet generation is a powerful technology in which one immiscible fluid (e.g. an aqueous solution) is dispersed in another (often an oil phase) to form highly uniform, picoliter- to nanoliter-sized droplets. These droplets serve as compartmentalized microreactors that can be produced in massive numbers, offering an elegant way to miniaturize and parallelize biological, chemical, and diagnostic assays [1]. The small volumes, large surface-area-to-volume ratio, and precise flow control possible in microchannels (e.g., Fig. 1) enable researchers to achieve faster reaction rates, reduce reagent usage, and maintain tight environmental control. This synergy has been integral to lab-on-a-chip systems used in fields spanning drug discovery, materials synthesis, and life sciences research [2].
In broad terms, microfluidic droplet generators can be grouped into two main categories:
- Passive generation: where no external energy is added beyond the pressure- or flow-driven forces that move fluids through channels (Fig. 2). In this approach, the relative flow rates of the two immiscible fluids control the droplet size, relying on phenomena such as capillary shear, channel geometry, and fluidic instabilities to break off droplets. Examples include:
- T-junction and Y-junction cross-flow geometries
- Co-flow geometries
- Flow-focusing devices
- Step emulsification and membrane emulsification
- Active generation: where an external actuation (e.g. electric, magnetic, acoustic, or mechanical valves) modulates droplet production in real time (e.g., Fig. 3). This additional energy can provide:
- More independent control over droplet volume and frequency
- On-demand droplets with precise timing
- Faster responses to changing experimental conditions
Whether passive or active, these devices typically operate under laminar flow regimes (low Reynolds numbers), so droplet pinch-off is dominated by the interplay of viscous shear forces, interfacial tension, channel confinement, and—in some designs—external fields such as electric or acoustic forces [2]. By carefully tuning these factors, highly monodisperse droplets (coefficient of variation typically below a few percent) can be formed at rates of hundreds to thousands of droplets per second (or more).
For simplicity, we will focus mainly on passively generated microdroplets in this technical note. The insights and instrumentation discussions below apply more generally to other forms of droplet generation, as well.
Relevance for Single-Cell Analysis and Beyond
A major impetus driving droplet microfluidics is the demand for single-cell research. Single cells exhibit intrinsic heterogeneity, meaning that even genetically identical cells can differ significantly in gene expression, metabolism, or drug responses [1, 4]. Traditional methods average signals over millions of cells, masking these subtle differences. Droplet-based microfluidics changes the paradigm by encapsulating individual cells into discrete droplets, effectively isolating them as tiny microreactors. This approach offers:
- High-throughput single-cell assays: By running thousands to millions of droplets in parallel, each containing exactly one cell, researchers can rapidly measure how cells respond to different reagents, test drug sensitivities, or perform sequencing.
- Quantitative genetics and proteomics: Microfluidic droplets have facilitated droplet-based PCR (dPCR) and digital assays for nucleic acids, enabling absolute quantification of DNA or RNA from single cells with minimal reagent consumption.
- Single-cell culturing and screening: Some droplet systems allow extended cell culture (e.g., water-in-oil droplets with specialized surfactants), enabling selection or screening of target phenotypes like antibody production or metabolic output [1].
Importantly, microfluidic droplet platforms can also handle sensitive cell lines (e.g., primary immune cells or rare tumor cells) by protecting them from cross-contamination or turbulent shear. The droplets remain stable if properly formulated with surfactants, effectively compartmentalizing each cell or cell cluster for experiments that range from short (minutes) to long (days) timescales.
Additionally, advanced droplet manipulations, such as droplet merging, sorting, and splitting, expand capabilities further. Researchers merge droplets containing different reagents or cell populations, sort droplets by fluorescence or other signals, and can even perform multi-step chemical reactions. This level of automation and reconfigurability is a hallmark of modern droplet microfluidic platforms and underscores their relevance to single-cell analysis [5].
Beyond interrogating and studying populations of single cells, this technology can also be applied to synthetic biology. Rapidly testing genetic circuits or combinatorial designs becomes easier when each droplet acts as a tiny “bioreactor.” Alternatively, libraries of enzymes or organisms can be compartmentalized in droplets, facilitating massively parallel selection or screening applications. Microfluidic droplet generators also facilitate compartmentalized chemistry setups, by performing thousands of miniaturized chemical reactions in parallel — thereby drastically reducing reagent usage and speeding up discovery.
Instrumentation Choices
Because the size and generation frequency of microdroplets are directly controlled by relative flow rates and channel geometry, precise fluid handling is paramount. There are three common instrumentation choices that researchers employ in this context: syringe pumps, pressure-based controllers, and peristaltic pumps.
Traditional methods generally use syringe pumps driving fluid into a microfluidic chip. Syringe pumps are relatively cheap and fairly simple, though they occasionally struggle with keeping extremely low rate flows steady, or with rapid changes in flow — both key considerations for droplet uniformity.
Most syringe pumps do not have backlash compensation built into their drive systems — which means they suffer from a dead zone in their stroke after each direction reversal (Fig. 4).
Moreover, at low flow rates, periodic stiction of the plunger as it rubs along the barrel’s inner surface causes the “slide-stick phenomenon” — resulting in jerky movements and noisy delivery rates. Discrete movements of the plunger at low flow rates due to the finite step size of the driving stepper motor are secondary, and can be mitigated by the use of micro-stepping on the motor, as well as choosing an appropriately small syringe volume.
Pressure-based controllers (offered by Fluigent, Elveflow, etc.) respond faster and achieve stable flows, but they are expensive (~$10K or more) and typically require a pressurized air or nitrogen source. Entire turnkey droplet platforms (e.g., Dolomite Microfluidics) can be tens of thousands of dollars and often lack versatility beyond droplet applications.
Without flow rate feedback, pressure-based control systems are highly sensitive to dynamic conditions within the microfluidic channel, such as to initial start-up oscillations and partial clogging by impurities. Velocity feedback makes these systems respond quickly to changing demands in flow (Fig. 5). They can even respond relatively quickly to dynamic changes in channel conditions, offering high stability for demanding droplet generation tasks. On the other hand, the setup requires a stable pressure source, and the corresponding hardware, including the source connections upstream of the controller, can be bulky.
Peristaltic pumps operate by compressing and releasing flexible tubing with rotating rollers, creating a “pumping” action that drives fluid forward. Because the fluid stays in the tubing and never touches the moving mechanical parts, the system is well-suited for handling sterile or shear-sensitive solutions. Although peristaltic pumps are typically simpler to operate in long-duration flow scenarios, their rolling motion causes a moderate pulsation in the flow. Users often add flow dampeners [8] or utilize a two-channel pump head with phase-offset rollers [9] to reduce flow instability when uniform droplets are desired.
In microfluidic droplet generation, peristaltic pumps are ideal in applications that need continuous and moderate flow control rather than sub-microliter accuracy. While peristaltic devices can reliably achieve consistent fluid delivery, their pulsatile flow profile can lead to slight variations in droplet size and frequency unless carefully managed. Nonetheless, they offer an accessible, low-cost, and robust solution for many droplet-based experiments, particularly where precise ultra-low flow rates are not critical and cell- or protein-friendly fluid handling is essential (Fig. 6).
Daisy Instruments for Microfluidic Droplet Generation
For microfluidic droplet generation that demands continuous, moderate-to-high volumetric flows (~10-1000 ul/min), the Daisy Peri P delivers affordable performance beyond typical peristaltic pumps. When combined with flow-dampening solutions, it can mitigate much of the pulsatility often associated with peristaltic pumping. This means that users benefit from the relatively gentle, shear-minimized operation of a peristaltic setup without sacrificing the fine precision necessary for reproducible droplet formation. The Daisy Peri P’s simple setup and robust engineering make it especially valuable for sensitive assays where controlled, near-constant flow is required — such as continuous perfusion experiments or droplet-based single-cell studies. Fig. 7 depicts a simple configuration that utilizes a single Daisy Peri P pump (with two channels), used in conjunction with pulsation dampers to drive a microdroplet generator.
For more stringent applications requiring exceptionally steady and smooth flow, the Daisy Piston stands out. Unlike classic syringe pumps that rely on an elastic seal at the top of a plunger dragging against the barrel, Daisy Piston avoids any slide-and-stick friction: the piston material is made of solid, hard PEEK, which moves within the barrel with a small clearance that avoids friction altogether. As such, the wear and tear is dramatically reduced, extending the lifetime of the piston over two orders, even compared to industrial syringe pumps. In fact, Daisy Piston is rated for up to 10 million dispensing cycles (typically, many years of operation) before servicing, providing 0.5% accuracy and less than 0.03% CV for full volume dispensing. With an integrated backlash compensation mechanism, it can automatically refill in mere seconds while maintaining flow continuity during pumping. Daisy Piston comes in three volume variants—100 µL, 1000 µL, and 5000 µL—to suit different fluid-handling needs. The 100 µL head in particular is ideal for microfluidic droplet generation, providing extremely uniform flow at low rates and effectively eliminating the stiction or re-priming disruptions that hamper typical syringe systems. Laboratories seeking stable continuous flows over extended durations can adopt Daisy Piston to achieve monodisperse droplets and precise reagent delivery without interruption. As an example, Fig. 8 depicts two Daisy Piston pumps that independently control the flow rates of the water and oil phases.
A major strength of the Daisy platform is its modular architecture, enabling straightforward integration of multiple fluid-handling modules. For complex droplet assays that demand multi-reagent or multi-step sequences, combining Daisy Piston (or Daisy Peri P) with Daisy Sol Val (solenoid manifold) or Daisy Multi Valve (rotary selector valve) offers huge flexibility. Users can rapidly and automatically switch between multiple input streams, or direct the output to various collection vials, all under synchronized software control (Fig. 9). This set-up is invaluable for multi-phase droplet generation, automated sample dilutions, or consecutive reagent additions within the same microfluidic workflow. Additionally, all these Daisy components boast high chemical compatibility — housing and fluidic paths use inert materials (PEEK, PTFE, or similar)—making them safe for a wide range of solvents, reagents, or biological samples without the risk of corrosion or contamination.
Coding Example: Microdroplet Generation with Daisy
Below is an example Python snippet using Daisy’s simple command structure for two Daisy Piston pumps and one Daisy Multi Valve, for the setup depicted in Fig. 9. Each pump aspirates a fixed volume of reagent (water- or oil-phase) quickly, then dispenses at target flow rates towards the corresponding inlet of the microfluidic device. The rotary valve module downstream of the microfluidic device switches to the waste vial before the start of each dispensing cycle — ensuring that the first few seconds of the run are not collected until the microdroplet stream stabilizes. The code waits for the pumps to finish before looping back to aspiration of fresh reagents into the pumps.
import time
# 1) Connect and name all Daisy modules
g0 = self.my_interface.groups[0] # first instrument chain is g0
pump_water = g0.inst[0] # first in chain is water pump
pump_oil = g0.inst[1] # second in chain is oil pump
outlet_valve = g0.inst[2] # last in chain is the valve
# 2) Home the Piston Pumps and the Multi Valve
pump_water.home()
pump_oil.home()
outlet_valve.home()
# 3) Repeatedly aspirate + dispense in a cycle for droplet flow
# (Here we do "strokes" that produce ~10 µL/min or ~50 µL/min average flow.)
# The Daisy Piston API: .run(<speed_in_ul_per_min>,
# <target_volume_position_in_ul>, <valve_num>, <waitBool>)
# valve_num = 0 => fluid in, valve_num = 1 => fluid out
# We are choosing a water pump with 100.0 ul capacity.
# The oil pump will have a 1000.0 ul capacity.
for cycle in range(10): # e.g. 10 cycles
# Aspirate water: 100.0 µL at 600 µL/min => takes 10 seconds
# Aspirate oil: 500.0 ul at 3000 ul/min => takes 10 seconds
pump_water.run(600.0, 100.0, 0, wait=False) # aspiration
pump_oil.run(3000.0, 500.0, 0, wait=True) # aspiration
# Dispense water at 10 ul/min and oil at 50 ul/min
# Will take about 10 mins
outlet_valve.select_port(1, wait=True) # to waste
pump_water.run(10.0, 0.0, 1, wait=False) # dispense all
pump_oil.run(50.0, 0.0, 1, wait=False) # dispense
time.sleep(4) # dispense for 4s # into waste
outlet_valve.select_port(2, wait=True) # to vial
pump_oil.run(50.0, 0.0, 1, wait=True) # cont. till doneKey Takeaways
Whether you are a PI aiming to incorporate droplet-based single-cell assays, or a biotech startup screening thousands of enzyme variants in droplet compartments, Daisy instruments offer a new paradigm: precision fluidic control in a compact, open-ended, and highly affordable format. Gone are the days of choosing between overpriced specialized droplet systems or clunky manual setups. Daisy’s modular approach means you can start small, grow as you need, and tackle droplet microfluidics (or many other fluidic tasks) with confidence and creativity.
With Daisy, you get:
1. Precision and Scalability: Daisy instruments deliver stable flows crucial for monodisperse droplet generation. You can fine-tune droplet size just by adjusting flow parameters in software.
2. Modular Automation: Each additional Daisy module that you add to your setup can increase capabilities exponentially. By adding a valve unit, for example, you can automatically switch between multiple reagents for complex droplet-based protocols (e.g., single-cell multi-conditions, synthetic biology screens).
3. Simplicity and Cost Savings: Daisy hardware is a fraction of the price of typical droplet platforms, and it requires minimal bench space.
4. Reconfigurability: Once you’ve finished a droplet experiment, you can reuse the same modules to do microplate dispensing, flow injections, or other fluid-handling tasks. No single-purpose “white elephant” device sitting idle, occupying precious bench space.
5. Easy Programming: Daisy’s unified software and AI “assistant” keep coding approachable — even for students or labs without a dedicated automation engineer.
For more details on Daisy modules (including motion stages, sensors, etc.), visit https://scalables.com, or reach out to request a demo at info@scalables.com.
We look forward to helping you implement your custom droplet generation workflows — and beyond!
References
[1] Li B, Ma X, Cheng J, Tian T, Guo J, Wang Y and Pang L (2023), Droplets microfluidics platform — A tool for single cell research. Front. Bioeng. Biotechnol. 11:1121870.
[2] Zhu P. and Wang L. (2017), Passive and active droplet generation with microfluidics: a review. Lab Chip, 17, 34-75.
[3] https://www.microfluidic-chipshop.com/
[4] De Jonghe J, Kaminski T.S., Morse D.B. et al. (2023), spinDrop: a droplet microfluidic platform to maximise single-cell sequencing information content. Nat Commun 14, 4788.
[5] Nan L., Mao T. & Shum H.C. (2023), Self-synchronization of reinjected droplets for high-efficiency droplet pairing and merging. Microsyst Nanoeng 9, 24 (2023).
[6] https://microfluidics-innovation-center.com
[8] For an example of a peristaltic pump pulsation damper, see https://leadfluid.com/pulsation-damper-for-peristaltic-pump/
[9] To see an example of a two-phase pump head usage, visit the application note at https://in.vwr.com/cms/tech-article-reduce-pulsation-double-drive-output-and-improve-accuracy-with-double-y-tube-sets
