Design and Synthesis of Fluorosurfactants for Microfluidic Droplet-Based Bioapplications

Abstract

Surfactants are central to droplet-based microfluidics, where they stabilize immiscible interfaces, prevent coalescence, and enable precise control of discrete fluid volumes for biological and chemical applications. Recent efforts in fluorosurfactant design have emphasized how variations in hydrophilic head groups, fluorinated tail architecture, and synthesis strategies determine performance in droplet systems. This review surveys advances from conventional polyethylene glycol (PEG)–perfluoropolyether (PFPE) architectures to emerging alternatives with nonionic polar head groups, highlighting those with the greatest potential to advance the field. Particular attention is given to reversible addition–fragmentation chain transfer (RAFT) polymerization, which has revolutionized fluorosurfactant development by enabling precise molecular control and tunable properties. This has allowed the integration of responsive functionalities, including temperature-sensitive N-isopropylacrylamide (NIPAM), pH-responsive 2-(dimethylamino)­ethyl acrylate (DMAEA), and zwitterionic monomers, thereby greatly expanding functional versatility. Such innovations improve droplet stability, suppress molecular exchange, and enhance biocompatibility under challenging experimental conditions. These advanced fluorosurfactants demonstrate exceptional utility across a spectrum of biological applications, including high-throughput screening, droplet digital PCR, single-cell genomics, cell encapsulation, and three-dimensional culture systems. Collectively, they establish a foundation for next-generation microfluidic technologies that demand robust compartmentalization with minimal biological interference.

1. Introduction

Over the past two decades, microfluidic droplet technology has emerged as a transformative tool in biological and biomedical research, enabling the generation of highly uniform, picoliter- to nanoliter-sized aqueous droplets dispersed in an immiscible oil phase. These compartmentalized systems serve as miniature reaction vessels, offering exceptional control over the cellular microenvironment, and facilitating applications ranging from single-cell analysis and synthetic biology to organoid culture and high-throughput drug screening.

Central to the success of droplet-based microfluidics is the use of surfactantsamphiphilic molecules that lower interfacial tension and stabilize droplet interfaces during formation, incubation, and downstream processing. Fluorinated surfactants, in particular, have become the gold standard for water-in-fluorocarbon oil emulsions due to their unique ability to maintain droplet stability without interfering with encapsulated biomolecules or cells. Their fluorophilic tails exhibit minimal interaction with hydrophobic or hydrophilic phases, allowing for biocompatibility and chemical inertness critical for sensitive bioassays.

Despite these advantages, the current generation of fluorosurfactants faces several limitations. Most commercially available surfactants are chemically inert and non-responsive, making droplet recovery or reagent exchange difficult without harsh demulsification methods, such as perfluorooctanol treatment. These procedures can compromise the viability of encapsulated cells and limit downstream applications, particularly in regenerative medicine, diagnostics, and cell therapy. Furthermore, limited tunability in surfactant structure constrains their use in more complex or dynamic biological systems that require on-demand release or environmental responsiveness.

To address these challenges, recent efforts have shifted toward the rational design and synthesis of “smart” fluorosurfactantsmolecular systems that combine the interfacial activity of conventional fluorinated surfactants with functional and responsive blocks capable of sensing or adapting to their surroundings. − By incorporating thermoresponsive, pH-sensitive, or bio-degradable segments into the surfactant structure, researchers can now control not only droplet formation but also disassembly and material release, offering new opportunities for dynamic control in droplet microfluidic systems. −

This Perspective outlines recent advances in the design, synthesis, and application of fluorosurfactants for droplet-based biological microfluidics (Figure ). We begin with a brief overview of microfluidic principles and the role of surfactants in droplet stabilization. We then focus on the modular design of fluorosurfactantsparticularly those synthesized via reversible addition–fragmentation chain-transfer (RAFT) polymerization in our groupand discuss how their tunable amphiphilicity enables reversible droplet stabilization. Finally, we highlight emerging applications in organoid engineering, drug screening, and synthetic biology, illustrating how these next-generation surfactants are poised to accelerate biological and medical translation.

1.

Visual summary of fluorosurfactant development for microfluidic droplet-based bioapplication.

2. Droplet-Based Microfluidics

2.1. Microfluidics

The microfluidic chip is a series of micro-channels etched or molded into materials (like siloxane and polydimethylsiloxane (PDMS)) for mixing, pumping, sorting, or controlling the biochemical environment. These chips offer excellent gas permeability, surface modification capabilities, and biocompatibility, making them invaluable for assessing cell viability in living organisms. The transparency of microfluidic chips also allows for easy observation of fluid dynamics using optical microscopes. Currently, soft lithography is the primary technique for fabricating microfluidic utilizing elastomer masks, stamps, and molds, known for its rapid prototyping advantages. Typically, photolithography is used to manufacture master molds for microfluidics, with a pre-polymer such as PDMS cured on top of the mold. Once cured, a PDMS-negative stamp of the mold is made and permanently bonded to glass (Figure ). ,

2.

Rapid prototyping using soft lithography. A soft lithography multistep process in which a master mold is created first, followed by curing a prepolymer substrate above, peeling it off, bonding it to a substrate, and punching access holes. Reproduced with permission from ref . Copyright 2017, Elsevier.

These microfluidic chips can achieve high-resolution features at the micrometer and even nanometer scales. PDMS, a widely used polymer in microfluidics, is favored for its biocompatibility, ready availability, transparency, hydrophobic properties, gas permeability, and elastomeric nature. As a result, microfluidic chips manufactured through soft lithography find extensive applications in life science. The application of microfluidics in biology and medicine has spawned several new lines of inquiry due to the field’s many benefits. Numerous uses have been shown, most of which have a biological emphasis. These include genetic engineering, proteomics, medical diagnostics, cell culture, drug research and development, and biosensors for biochemical and pathogen detection.

2.2. Droplet-Based Microfluidics

Microfluidic reactors are becoming increasingly popular as a platform on which to conduct biological and chemical research, and this has spurred substantial advancements in the production of water-in-oil emulsions within these settings. The interfacial instability that exists between the two immiscible phases is the primary cause of the droplet formation process. During this process, the interface between the continuous and dispersed phases is distorted, which ultimately results in the pinching off the dispersed phase as well as the production of droplets. Droplet-based microfluidics is better capable of high-throughput creation of monodisperse droplets as compared to the standard droplet generation techniques. The objective of droplet-based microfluidics is the creation and manipulation of discrete quantities of immiscible fluids. Droplet microfluidics offer a variety of applications, including medication administration, emulsification, nano/micro-particles creation, and cell encapsulation.

In microfluidics, the passive and active techniques are the two primary ways that may be used to achieve the desired state of droplet creation. This state can be established in terms of droplet size and production rate. The operation of the passive techniques is predicated on the geometry of the channel and the physical characteristics of the immiscible phases. On the other hand, active approaches manage the dynamics of droplet breakdown by making use of the force fields that are external to the droplets. Changing the injection rates or drive pressures of the dispersed and continuous phases into the microfluidic device is a common passive technique for manipulating droplets. The use of surfactants is one more method that may be used to change the droplet size as well as the droplet formation frequency.

This review aims to present a concise overview of the synthesis methods of diverse fluorosurfactant classes developed during the past decade, highlighting variations in headgroup structure, miscellaneous polar head, along with their potential applications. The review is organized into several sections: we begin by examining surfactant selection criteria, followed by an analysis of fluorosurfactants and their applications specifically in fluorinated oil environments. The final section explores potential biological applications of these surfactants.

3. Principles of Fluorosurfactant Design

3.1. Amphiphilicity and Interfacial Tension Control

Surfactants derive their unique functionality from amphiphilicitypossessing both hydrophilic (water-loving) and hydrophobic/fluorophilic (water-repelling/fluorine-attracting) regions within one molecule. This structural duality enables them to naturally accumulate at boundaries between oil and water or fluorinated oil and water, thereby decreasing interfacial tension and enhancing stability of dispersed phases in microfluidic droplet systems. This property is fundamental to controlling droplet characteristics essential for applications ranging from single-cell analysis to digital PCR and drug screening platforms.

3.1.1. Mechanism of Interfacial Tension Reduction

The phenomenon of interfacial tension results from unbalanced cohesive forces where two immiscible liquids meet. Surfactants minimize this tension by congregating at interfaces and arranging themselves strategicallytheir hydrophilic heads facing the aqueous environment while their hydrophobic or fluorophilic tails extend into the oil phase. This strategic arrangement disrupts surface molecular forces, lowering the energy required to maintain the boundary and consequently reducing interfacial tension.

Fluorosurfactants feature distinctive fluorophilic tails with minimal polarizability and robust carbon–fluorine bonds that enhance dissolution in fluorinated oils. These properties facilitate substantial reduction in interfacial tension, which is essential for creating stable water-in-oil emulsions that resist molecular exchange and droplet merging.

3.1.2. Factors Influencing Interfacial Tension Control

The effectiveness of reducing interfacial tension depends on a number of factors. The hydrophilic-lipophilic balance (HLB), which denotes the equilibrium between hydrophilic (polar) and lipophilic (nonpolar) groups in emulsifier molecules, quantified by the HLB number. This number signifies a surfactant’s capacity to stabilize oil-in-water or water-in-oil emulsions. Besides, the capacity to create stable interfaces are greatly impacted by the selection of the hydrophilic head group (such as carboxyl, sulfate, phosphate, or zwitterionic). , Ionic and zwitterionic groups can improve droplet stability through electrostatic interactions, while nonionic head groups (like PEG) frequently offer superior biocompatibility. The concentration of surfactant is also important, as the critical micelle concentration (CMC) is the the primary chemical-physical parameter for characterizing pure surfactants in terms of surface activity and self-assembled aggregation. It is the threshold above which surfactants form micelles, significantly reducing surface tension. Fluorosurfactants typically have a lower CMC than hydrocarbon-based surfactants, which reflects their high surface activity. Furthermore, some fluorosurfactants include responsive segments (polar head)­(e.g., poly­(N-isopropylacrylamide) (pNIPAM), 2-(dimethylamino)­ethyl acrylate (DMAEA) that can modulate interfacial tension in response to environmental triggers, enabling controlled droplet destabilization or molecular release, adding a layer of functional versatility to the surfactant design. ,

3.2. Molecular Architecture (e.g., Block Copolymers with PFPE Tails and Hydrophilic Heads)

The creation of novel surfactant molecules and the study of their properties have allowed for recent advancements in the applications of droplet-based microfluidics. Surfactants are molecules that are both hydrophilic and amphiphilic, and they are typically added to either the continuous or the dispersed phase to avoid droplet coalescence (Figure ).

3.

Schematic diagram of droplet generation and surfactants absorbed to the interface. Biologic materials (DNA, RNA, proteins) and/or organisms may be encapsulated in water-in-oil emulsion drops. Microfluidic devices use a flow focusing geometry to create drops (see image on the left). A layer of surfactants forms at the contact as seen at the right (arrow 1). Surfactant layers stabilize emulsions and prevent biomolecules and cells from adhering to the interface (arrows 2 and 3). Functional drop-based biological experiments need fine-tuning the surfactant’s molecular structure and content. Reproduced with permission from ref . Copyright 2008 Royal Society of Chemistry.

In droplet-based microfluidics, surfactants are essential for droplet stability and preventing droplet coalescence during incubation due to their ability to absorb at the oil-water interface. They contribute to droplet interface stability, system biocompatibility, and the molecular exchange that occurs between droplets. As a result, surfactants are crucial for maintaining droplet stability in a microfluidic micro-bioreactor.

Biocompatibility, stability, and molecular exchange are all factors to consider when choosing a surfactant. The surfactant should be biocompatible so that it does not hinder enzyme function or cell viability inside the droplets. Droplets used as bioreactors must be stable. The stability of the droplets is crucial to the success of a digital polymerase chain reaction (PCR) process based on droplets, and the surfactant plays a key role in this. It is important to remember that the droplet is not a completely airtight container. Over time, droplets that are physically near to one another may trade small molecules with one another. This process may be successfully inhibited by selecting the appropriate oil and surfactant combinations. Surfactants have an influence on the process of phase partitioning, despite the fact that the usage of fluorinated oils reduces the amount of molecular exchange that occurs during phase partitioning. During thermocycling, surfactant bilayers may develop between droplets and can be permeable to some molecules, particularly tiny molecules. As proved by mineral oil and nonionic surfactant, this transport process is a function of the lipophobicity of the molecules to be carried.

Since the hydrophobic group of the surfactant molecules extends into the oil, the oil phase is also a critical consideration in the surfactant selection process. In PDMS-based systems, the variety of bio-chemical tests is restricted due to the incompatibility of silicone oils with microfluidic chips. , The two most common kinds of continuous phase for water-in-oil (w/o) droplet emulsion are hydrocarbon oils and fluorocarbon oils.

Hydrocarbon oils, which have been employed effectively in PCR and guided evolution of enzymes, provide the basis of emulsion PCR. However, hydrophobic chemicals can phase partition into oil in hydrocarbon systems, reducing their usage. Single-gene amplification may also be performed in fluorinated oils and fluorosurfactants. ,,

Hexadecane is the most widely used hydrocarbon, and its related surfactants include Span 80, Tween 20/80, etc. Due to their high gas permeability and incompatibility with non-fluorinated molecules, fluorocarbon oils are commonly employed in cell culture, enzyme reactions, and PCR.

3.3. Fluorosurfactants and their Applications in Fluorinated Oil

Surfactant molecules need to provide a strong droplet interface to ensure the droplets remain stable throughout time. Surfactants with fluorus tail groups in the C6 to C10 range do not provide sufficient long-term emulsion stability for widespread use in microfluidic devices. Therefore, long-chain perfluoropolyethers (PFPEs) that are readily accessible on the market and provide stability even for 200–220 μm droplets.

Using fluorinated oils and fluorosurfactants is crucial to many droplet-based microfluidics applications (Table ). The popularity of fluorinated oils stems mostly from two factors. First, they are desirable because they are insoluble in water and most organic molecules. Organic and silicone oils’ exchange constraints are resolved since the chemicals contained in the droplet shouldn’t phase partition and should instead stay in the droplet. Fluorinated oils have a secondary benefit of being biocompatible.

1. Microfluidic Surfactants Used for Emulsions of Water and Fluorinated Oil.

Surfactant	Source	Application	Fluorinated oil
PFPE-COONH4	Krytox, Dupont	Multiple emulsions, Cells in droplets	Novec 7500, FC-40
PFPE-COOH	Krytox, Dupont	Droplet detection	Novec 7500
PFPE-DMP	Krytox, Dupont	Cells in droplets, Bacteria in droplets	Novec 7500, FC-40
PEG-PFPE2 (EA surfactant)	RAN Biotechnologies	Cells in droplets, Single-cell genome sequencing, Yeasts in droplets, PCR, Single-molecular genomic screening (ddPCR)	Novec 7500, FC-40
Jeffamine-PFPE2 (Pico-surf)	Sphere Fluidics	Cells in droplets, Bacteria in droplets, Ultra-high throughput screening platform, PCR	Novec 7500, FC-40
PFPE-Tris	Homemade	Cells in droplets, DNA polyplex synthesis, Enzymatic activity assay	Novec 7500
PFPE-dTG	Homemade	Cells in droplets, PCR, Drug diffusion test	Novec 7500
LPG(OMe)-PFPE2	Homemade	Dye diffusion test	Novec 7500
PFPE-ITG	Homemade	Dye diffusion test	Novec 7500
FSHPEG900HA	Homemade	3D Printing of Capsules	Novec7500, Novec 7100
Zonyl FS300	Homemade	Janus emulsion assay	FC770
PEG5PFPE1	Homemade	Mechanical force measurements in zebrafish	Novec 7700
PFPE-pNIPAM	Homemade	Cell-laden microgels	Novec 7100
KryAz600	Homemade	Light-driven merging of droplets	Novec 7500
P(2-HEA)4-PFPE	Homemade	Cells in droplets, Yeasts in droplets, PCR, Dye diffusion test	Novec 7500
Surfactants 1b, 2, 3, 4, and 5	Homemade	Fishing of Biomolecules, Capsule Fabrication, Single-cell gene expression	Novec 7500

3M offers a few fluorocarbons for sale, including Perfluoro-compound (FC)-40, FC-77, hydrofluoroether (HFE) 7500, and HFE 7100. Several different surfactants, including Krytox (DuPont), dimorpholinophosphinate perfluoropolyether (DMP-PFPE), block copolymer of poly-ethylene glycol and perfluoropolyether (PEG-PFPE), block copolymer of linear polyglycerol and perfluoropolyether (LPG-PFPE), and fluorinated nanoparticles (F-NPs), have been developed for use. Since PEG-PFPE is biocompatible and has a stabilising effect, it is the most popular of these surfactants.

3.3.1. PEG-Based Di- and Tri-Block surfactants

It’s crucial to acknowledge that ionic interactions can enable organic molecules in the aqueous phase to migrate into the continuous fluorous oil phase, thereby posing challenges to the compartmentalization process and the downsizing endeavor’s viability. Additionally, ionic interactions between surfactant and protein molecules may disrupt the native structures and functions of biomolecules, especially proteins. To address these issues, Holtze et al. have proposed the use of PEG (Mn 600)-coupled PFPEs with a triblock PFPE-PEG-PFPE structure (Figure ), Commonly known as EA surfactant. Their findings illustrate swift surfactant diffusion to the droplet interface, long-term emulsion stability, and a nonionic droplet interface. During synthesis a homo-bifunctional PEG head is connected to two Krytox molecules through an amide bond to form a triblock copolymer surfactant. However, this synthesis proved challenging to reproduce due to insufficient characterization and the use of starting ingredients that are not readily accessible from commercial sources.

4.

Reaction scheme of a triblock copolymer surfactant (PFPE-PEG-PFPE). Reproduced with permission from ref . Copyright 2008 Royal Society of Chemistry.

To overcome shortcomings of the Holtze study, Matochko et al. describe a straightforward three-step process for synthesizing a surfactant from readily accessible ingredients (Figure ). Deprotection and the Mitsunobu reaction turned polythene glycol into diamine derivative 2. The acyl chloride derivative of Krytox 3 reacted with an excess of diamine 2 to generate surfactant 4 (PFPE-PEG-PFPE) in high quantities. Through crystallization, the solid intermediates may be isolated and refined. However, the tetrachlorophthalimide (TCP) intermediate 1 proved challenging to crystallize throughout this synthesis.

5.

Reaction scheme of surfactant 4 (PFPE-PEG-PFPE). Reproduced with permission from ref . Copyright 2012, Elsevier

Furthermore, Zinchenko et al. have reported a structurally similar derivative of the surfactants previously described by Holtze and Matochko, utilizing Jeffamine ED-600 as the head group and two PFPE units as tail groups (Figure ). Jeffamine ED-600, a polyether diamine with a molecular weight of 600 g/mol containing a PEG backbone, contributes to the synthesis of PFPE-Jeffamine-PFPE surfactant, marketed as Pico-Surf (Dolomite Microfluidics, Royston, UK).

6.

Reaction scheme for the synthesis of surfactant PFPE-Jeffamine-PFPE.

In summary, the fluorosurfactants under discussion are synthesized through multi-step processes that circumvent the need for product purification, as seen in PFPE-PEG and PFPE-Jeffamine-PEG. Consequently, the final mixture necessitates separation via centrifugation or filtration to recover the desired reaction product. Regrettably, this approach often results in less consistent outcomes in terms of the synthesis process and the characteristics of the final product. As a result, the solubility in fluorous oils is suboptimal, and the materials exhibit reduced biocompatibility in comparison to initial expectations.

3.3.2. Miscellaneous Polar Head-Based Di- and Tri-Block Surfactants

While PEG has long been considered biologically inert, recent research findings challenge this notion by suggesting that PEG’s hydrophobic properties can modulate the bioactivity of specific proteins. Consequently, the suitability of PEG-based surfactants for compartmentalizing biomolecules in droplet-based biological affinity tests reliant on hydrophobic protein interactions has come into question. Consequently, researchers have explored the development of di- and tri-block fluorosurfactants featuring varied polar head groups, both short and long-chain fluorinated tails. Chiu et al. conducted a study revealing that fluorosurfactants incorporating tris head groups, characterized by three hydroxy groups, have the capability to encapsulate mammalian cells within water-in-oil-in-water (w/o/w) double emulsions (Figure a). Extended incubation of these double emulsions containing mammalian cells resulted in polydisperse droplet size distributions, whereas short-term incubation of single emulsions containing bacterial cells exhibited excellent performance. Notably, their study did not provide insights into the release dynamics of the enclosed cells from the inner droplets of the double emulsions. Wagner et al. introduced linear polyglycerol-based tri-block copolymer fluorosurfactants, characterized by 14 repeating units, each featuring hydroxy or methoxy pendant groups, mirroring the length and geometry of the highly successful PEG600-based tri-block copolymer fluorosurfactants (Figure b). Moreover, it is worth noting that surfactants with methoxy groups tend to exhibit greater stability compared to their hydroxy group counterparts, a feature attributed to their polarity.

7.

Polar head group diversity in fluorosurfactants. (a) PFPE-Tris was produced in two steps: (1) PFPE-COOH to PFPE-COCl and (2) PFPE-COCl to PFPE-Tris. (b) Two perfluoropolyether “PFPE₂” blocks flank a central linear polyglycerol “LPG­(OH)” or poly­(methyl glycerol) “LPG­(OMe)” in the chemical structure of polyglycerol-based triblock surfactants. Reproduced with permission from ref . Copyright 2016 Royal Society of Chemistry.

In conclusion, the limited selection of nonionic fluorosurfactants, each possessing distinct polar head groups, underscores the significant influence of polar group geometry and type on fluorosurfactant performance within the realm of droplet microfluidics, surpassing the impact of highly polar hydrophilic head groups.

3.3.3. Tailored Surfactants Synthesized via Reversible Addition–Fragmentation Chain Transfer Polymerization

For biocompatibility, surfactants should have hydrophilic head groups that don’t disrupt biological or chemical processes within droplets. This has led to the use of biocompatible components like PEG in droplet-based microfluidics to reduce interference. However, PEG-based surfactants synthesized by reacting PFPE with PEG often involve complicated synthesis methods with low yields. These complex processes can create unwanted byproducts, including di-block structures, unmodified precursors, and ionically bonded surfactant molecules.

Building on the novel RAFT polymerization, Li et al. synthesized a series of PFPE-based dendronized fluorosurfactants utilizing three distinct hydrophilic monomers (polar head): oligoethylene glycol acrylate (OEGA), 2-hydroxyethyl acrylate (2-HEA), and 2-(methylsulfonyl)­ethyl acrylate (MSEA) (Figure ). As detail, 2-(butylthiocarbonothioylthio)­propionic acid (BTPA)-PFPE macro-chain transfer agent (macro-CTA) was first synthesized via an esterification coupling reaction employing N-(3-(dimethylamino)­propyl)-N′-ethylcarbodiimide hydrochloride/4-(dimethylamino)­pyridine (EDC/DMAP) as the coupling system. Subsequently, the polymerization of monomers was carried out through a one-step RAFT polymerization using azoisobutyronitrile (AIBN) as the initiator. This controlled living radical polymerization technique allows for precise regulation of fluorosurfactants’ molecular composition, particularly the critical balance between hydrophilic and hydrophobic properties. As a result, these surfactants form consistent, densely packed monomolecular layers on droplet surfaces that effectively minimize molecular interactions between droplets and prevent aggregation, even under high-temperature conditionsaddressing the thermal stability limitations of conventional surfactants and expanding the potential applications in biological systems requiring robust droplet integrity.

8.

Synthesis of fluorosurfactants using novel RAFT polymerization.

3.3.4. Emerging Smart Fluorosurfactants

Various functional fluorosurfactants have been developed for diverse applications. For instance, An et al. linked PFPE with thermally responsive polymerseither pNIPAM or poly­(2-ethyl-2-oxazoline) (pEtOx). Their research demonstrated that these surfactants could stabilize emulsions at temperatures below their lower critical solution temperature (LCST), but would trigger demulsification when heated above this threshold, primarily due to the increased hydrophobicity of pNIPAM or pEtOx in solution. In more recent work, Chowdhury et al. paired a PFPE tail with various oxidation-responsive polar head groups to create a versatile parent surfactant capable of generating multiple multifunctional variants. Incorporating functional components into fluorosurfactants enables the creation of multifunctional products, expanding potential applications across adsorption, bioanalytics, catalysis, formulations, coatings, and programmable emulsion subsets.

Apart from the previous synthesized various fluorinated surfactants from Li et al., Cheng et al. have synthesized other functional fluorinated surfactants for water-in-oil droplet formation via RAFT polymerization. Thay have selected three RAFT agentsBTPA, 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid (CPADB), and 2-(dodecylthiocarbonothioylthio)-2-methylpropionic acid (DDMAT)based on the targeted (meth)­acrylate monomers (Figure a). These monomers (polar head) included 2-HEA, pH-responsive DMAEA, temperature-responsive NIPAM, and zwitterionic monomers such as (2-(methacryloyloxy)­ethyl)­dimethyl-(3-sulfopropyl) ammonium hydroxide (SBMA), 2-methacryloyloxyethyl phosphorylcholine (MPC), and 3-(2-methacryloyloxy)­ethyl)­dimethyl ammonio)­propionate (CBMA) (Figure b). , The product yield of these RAFT synthesized derivates was relatively high around 90 %, and complied with the narrow molecular weight distribution (Đ ≤1.10). ,,

9.

Synthesis of fluorosurfactants. (a) Reaction pathway for creating PFPE Macro-RAFT Agents and (b) various functional monomers employed in fluorosurfactant production. Reproduced with permission from ref . Copyright 2024 American Chemical Society.

4. Fluorosurfactant for Biological Applications

Droplet microfluidics supports a broad range of biological applications, including high-throughput screening, single-cell genomics and transcriptomics analysis, droplet digital PCR, direct microbial evolution, and the formation of cell spheroids through either cell aggregation in confined microenvironments or encapsulation within microgels (Figure ).

10.

Schematics illustrating fluorosurfactant for biological applications.

4.1. Nucleic Acid Amplification

Microfluidic devices are highly adaptable, allowing for precise and rapid manipulation of very small amounts of fluid. Therefore, microfluidic technologies have the potential to boost the throughput of DNA analysis. Single-cell genomics, which plays a crucial role in understanding individual cell activity throughout normal development and illness, has drawn increased interest in the research of droplet-based microfluidic devices. DNA is a kind of nucleic acid that contains the genetic blueprint for all forms of cellular life. Therefore, numerous disciplines are interested in DNA characterisation, including clinical diagnostics and pharmacological research. Beer et al. were the first to conduct PCR using a droplet-based microfluidic technology. Since then, droplet microfluidic platforms have been employed for DNA extraction and sequencing.

In addition to DNA analysis, droplet-based microfluidic devices have significantly advanced RNA analysis, particularly in the field of single-cell RNA sequencing (scRNA-seq). The development of ultra-high-throughput single-cell RNA-seq platforms has demonstrated considerable potential in characterizing complex biological systems and even entire organisms. Recently, Zhang et al. have emerged three prevalent droplet-based systems as robust platforms for high-throughput scRNA-seq: inDrop, Drop-seq, and 10X Genomics Chromium. These droplet-based microfluidic systems enable rapid compartmentalization and encapsulation at rates reaching tens of thousands of droplets per second, allowing for the scalable generation of millions of nanoliter-volume droplets suitable for single-cell reactions.

4.2. Cell Culture in Microfluidic

Microfluidic cell culture is characterized technically by its strong spatiotemporal control over the cellular microenvironment and its numerous means of analysis, including microscopy, electrophysiological, and outlet media investigations. Automatic operation is possible, and it requires fewer medium changes and less medium evaporation than conventional methods. Because there are fewer cells to care for, less of the supporting medium and nutrients are needed. Modelling physiological conditions and gaining fundamental insights into intercellular processes are not the sole applications for microfluidic cell culture research. Because they provide precise control over the modelled processes, they are also an appealing method for gaining insight into diseases.

4.2.1. Single Cell Encapsulation

As synthetic biology and biotechnology continue to advance rapidly, high-throughput screening of gene functionality and engineered cells or strains is becoming increasingly important. Recent developments in automated microfluidic devices further assist in realizing the promise of droplet technology for high-throughput screening by allowing the compartmentalization of single cells in w/o emulsion droplets. However, aqueous phase analysis is not applicable to these single-emulsion droplets, and there is little communication between the droplet’s internal environment and the exterior phase. To get over these constraints, Chan et al. describe a microfluidics-generated w/o/w double emulsion (DE) droplet-based screening tool for gene amplification on chips. Their finding substantiates previous research suggesting that cultivating bacteria inside droplets facilitates not only bacterial separation but also signal amplification from a single bacterium, allowing for the pre- and post-error correction screening of functionally-correct genes produced by a microarray gene synthesizer. Moreover, microfluidics-based high-throughput screening plays a critical role in strain engineering by enabling the efficient evaluation of large strain libraries. For instance, a high-throughput G protein-coupled receptor (GPCR)-based autocrine screening system was developed using the microfluidic Xdrop platform with DE20 cartridges to facilitate the production of secondary metabolites in yeast Saccharomyces cerevisiae. This system enabled the identification of high-yielding strains capable of producing valuable aromatic compounds, including tryptamine and serotonin.

4.2.2. Multicellular Spheroids Encapsulation

Using multicellular spheroids as microtissues is a promising approach to tissue engineering, and this is especially true for stem cell spheroids. In addition to lacking the ability to supplement signals from extracellular matrix (ECM) (e.g., hydrogel) for improved differentiation, conventional ways of manufacture spheroids are hampered by limited throughput and polydispersity in size.

Many cellular properties may be adjusted when cells are grown in a three-dimensional (3D) spheroid culture. Enhanced production of functional indicators in normal cells has been shown to result from cell–cell interactions and 3D mechanical and molecular gradients (e.g., oxygen, nutrition). Examples include the increased expression of functional indicators like alpha-fetoprotein (AFP) or cytochrome P450 (CYP450) in hepatocytes when they are cultured as spheroids.Furthermore, cancer cells (e.g., Henrietta Lacks (HeLa), human embryonic kidney (HEK)) and stem cells (e.g., human mesenchymal stem cells (hMSCs)) have shown that spheroid culture typically promotes a metabolic shift towards higher glycolysis. Using oil-isolated droplets, which are significantly more susceptible to nutrient depletion and by-product build-up than standard culture containers, necessitates a detailed assessment of the cellular biochemical milieu, as shown by this finding. Chan et al. described the use of pico-liter sized bioreactors comprised of microfluidics-generated w/o/w DE droplets for fast cell assembly and a well-controlled microenvironment for spheroid development.

5. Challenges and Future Perspectives

Despite the remarkable progress in the design and application of fluorosurfactants for droplet-based microfluidics, several challenges remain that must be addressed to enable broader clinical and commercial translation. These include ensuring regulatory compliance, improving biocompatibility, and designing sustainable alternatives in light of growing environmental concerns.

5.1. Integration with GMP-Compatible Workflows

One of the key bottlenecks for the clinical translation of droplet microfluidic systems is the lack of integration with good manufacturing practice (GMP)-compliant processes. The materials and surfactants commonly used in academic research are not always approved for human use or scalable for industrial production. Fluorosurfactants, while effective in stabilizing water-in-oil droplets, must be manufactured under stringent quality controls to meet regulatory standards for safety, purity, and traceability.

To address this, future work must focus on: (1) Standardizing synthesis and purification protocols for biomedical-grade fluorosurfactants. (2) Developing scalable, reproducible microfluidic platforms that minimize batch-to-batch variation. (3) Creating closed, automated systems to ensure sterility and minimize contamination risks during organoid production and cell encapsulation.

5.2. Strategies for Clinical Translation

For fluorosurfactant-enabled microfluidics to reach clinical settings, several translational hurdles must be overcome:

5.2.1. Biocompatibility and Regulatory Clearance

Regulatory approval for clinical or therapeutic use requires extensive toxicological evaluation. The surfactants must be non-immunogenic, biodegradable, and safe for in vivo or ex vivo applications such as cell therapy or diagnostics.

5.2.2. Nontoxic Droplet Recovery

A major limitation of current systems is the reliance on chemical demulsifiers such as perfluorooctanol (PFO) for droplet breaking, which can damage cells and interfere with downstream applications. Our group’s recent development of thermoresponsive fluorosurfactants provides a non-invasive alternative by enabling temperature-triggered droplet destabilization without toxic reagents.

5.3.3. Environmental Responsibility

The growing regulatory scrutiny of per- and polyfluoroalkyl substances (PFAS) has justifiably raised concerns regarding the persistence and potential toxicity of certain long-chain fluorinated compounds. However, it is essential to distinguish between different classes of PFAS. PFPE-based surfactants, particularly PFPE-PEG-PFPE triblock copolymers, are indispensable in droplet-based microfluidics due to their ability to form stable, biocompatible emulsions essential for high-throughput biological assays and single-cell analyses. Notably, there is no significant evidence of toxicity associated with PFPE-based surfactants under typical laboratory or biomedical usage conditions.

These surfactants have been extensively validated in biological workflows, including digital PCR, enzyme kinetics, single-cell encapsulation, and droplet-based screening systems. Furthermore, compared to legacy PFAS compounds, PFPEs are chemically inert, thermally stable, and have low volatility, which reduces their environmental mobility and bioaccumulation potential.

Recognizing both the critical role of PFPE surfactants and the evolving regulatory landscape, our research group supports a balanced, evidence-based approach to PFAS governance. From a regulatory perspective, we advocate for structure- and use-specific risk assessments rather than blanket bans, aligning with organisation for economic co-operation and development (OECD)’s call for subclass-based management of PFAS. PFPEs, by virtue of their chemical inertness, low volatility, and contained use, represent a low-risk subclass that should be regulated accordingly.

We also promote proactive stewardship by ensuring proper containment and disposal, sharing toxicity and lifecycle data, and contributing to policy discussions where scientific input is critical. As part of our commitment to environmental sustainability, we are concurrently developing partially fluorinated and environmentally responsive surfactants that retain the high performance of PFPE-based systems while minimizing ecological impact. By combining responsible material selection, performance-driven design, and active engagement with regulatory frameworks, we aim to support both technological innovation and long-term environmental responsibility.

5.3. Opportunities for AI-Guided Surfactant Design and Simulation-Based Interfacial Tuning

Recent advancements in computational chemistry and machine learning offer powerful new tools for the design of next-generation fluorosurfactants. In silico models can now predict interfacial behavior, droplet stability, and surfactant assembly dynamics, enabling rational design with fewer trial-and-error iterations.

5.3.1. AI-Driven Molecular Design

Data-driven approaches can identify novel amphiphilic architectures with desired responsiveness and biocompatibility. By training models on large datasets of surfactant–interface interactions, researchers can accelerate the discovery of tailored surfactants for specific cell types or biological applications.

5.3.2. Molecular Dynamics Simulations

Atomistic and coarse-grained simulations can elucidate surfactant packing, interface stabilization, and micelle behavior under physiological conditions. These insights can guide the structural tuning of block copolymers or facilitate the integration of stimuli-responsive segments.

By embracing these computational strategies, the field can move toward the predictive design of “smart” surfactants that meet application-specific requirements across a wide range of biomedical scenariosfrom organoid engineering to in situ therapeutic delivery.

Droplet-based microfluidics has emerged as a powerful platform for diverse biological and chemical applications, with fluorosurfactants playing a critical role in maintaining droplet stability and functionality. As highlighted in this review, significant advancements have been made in fluorosurfactant development, moving from traditional PEG-PFPE architectures to sophisticated designs with customizable properties through RAFT polymerization and alternative polar head groups.

The evolution of fluorosurfactants has addressed key challenges in droplet microfluidics, particularly biocompatibility, thermal stability, and prevention of molecular exchange between droplets. PEG-based surfactants, while widely used, have shown limitations in synthesis reproducibility and potential interference with protein functions. This has driven the development of alternative designs with varied polar head groups such as Tris, linear polyglycerol, and dendronized architectures that offer improved performance under specific conditions.

Perhaps the most significant breakthrough has been the application of RAFT polymerization to fluorosurfactant synthesis, enabling unprecedented control over molecular structure and composition. This approach has facilitated the incorporation of functional elements including thermal-responsive NIPAM, pH-responsive DMAEA, and biocompatible zwitterionic monomers, expanding the toolkit available for specialized applications. These advances have directly contributed to improved performance in nucleic acid amplification, single-cell analysis, and three-dimensional cell culture systems.

Despite these advancements, several challenges remain to be addressed. Current fluorosurfactants still face limitations in maintaining stability under extreme conditions like high temperature cycling in PCR applications. Additionally, molecular exchange between droplets remains problematic for certain small molecules despite improved surfactant designs. The biocompatibility of surfactants with sensitive biological systems such as primary cells and embryos requires further optimization to minimize interference with cellular functions.

Looking forward, the field would benefit from the development of ’’smart’’ fluorosurfactants with programmable responsiveness to specific stimuli, allowing dynamic control over droplet properties. Integration of additional functionalities such as targeted binding capabilities, controlled permeability, and enhanced optical properties could expand applications in sensing and diagnostics. Simplification of synthesis routes for advanced fluorosurfactants would also facilitate broader adoption, as current methods often require specialized expertise and equipment.

The commercialization of novel fluorosurfactants represents another important frontier, as the majority of advanced designs remain confined to academic research. Standardization of performance metrics and comprehensive biocompatibility testing will be essential for translating these materials to clinical and industrial applications. Additionally, environmental considerations warrant attention, with a focus on developing fluorosurfactants with reduced environmental persistence while maintaining performance.

As droplet microfluidics continues to advance toward clinical applications in diagnostics and therapeutics, the development of fluorosurfactants that meet regulatory requirements while providing robust performance under diverse conditions will be increasingly important. The convergence of fluorosurfactant design with advances in microfluidic device fabrication promises to open new avenues for integrated systems with enhanced capabilities for single-cell analysis, therapeutic development, and synthetic biology applications.

R.Q. conceived the idea and supervised this project. R.Q. and X.L. wrote the paper with input from all authors. R.Q. and T.P.D. provided critical feedback and helped revise this article.

The authors declare no competing financial interest.