Membrane Fusion-Inspired Nanomaterials: Emerging Strategies for Infectious Disease and Cancer Diagnostics.

1
Introduction
Membrane fusion is a fundamental biological process[

1

] that underpins various cellular functions, such as intracellular trafficking,[

2

] neurotransmitter release,[

3

] and viral infection.[

4

] This intricate and highly regulated mechanism involves the merging of two separate lipid bilayers into a single continuous membrane, facilitating the exchange of contents between cellular compartments or between cells and their external environment.[

5

] Understanding the molecular principles of membrane fusion has provided critical insights into cellular physiology and inspired the development of innovative nanomaterials that mimic these natural processes for biomedical applications.[

6

]

The design of membrane fusion‐inspired nanomaterials is deeply rooted in the molecular understanding of biological fusion mechanisms. Key players in this process include proteins such as soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs)[

7

] and viral fusion proteins.[

4
,
8

] These elements lower the energy barriers required for membrane merging and stabilize transient fusion intermediates, such as stalk and hemifusion states, which are critical for successful content mixing. Additionally, lipid molecules, such as cholesterol and specific phospholipids, contribute to membrane flexibility and structural rearrangements, further facilitating the fusion process.[

9

]

Building on these molecular insights, recent advancements in nanotechnology have enabled the engineering of liposomes, exosome‐mimicking nanoparticles, and other lipid‐based systems that closely replicate natural membrane fusion processes.[

10
,
11

] Notably, these nanosystems can be functionalized with ligands, fusion peptides, or DNA‐based triggers to increase their targeting specificity and responsiveness to environmental cues.[

12

] Such functionalization approaches align with broader strategies in the development of surface‐modified nanomaterials aimed at enhancing therapeutic and diagnostic performance for biomedical applications. This involves the incorporation of small molecules, peptides, proteins, or nucleic acids to impart novel recognition or reactive functionalities, along with chemical modifiers or polymers that improve stability and biocompatibility.[

13

] As a result, the practical applications of these membrane fusion‐inspired nanomaterials are increasingly evident in their potential to revolutionize diagnostic methods for cancer, viral infections, and other diseases.[

14
,
15
,
16

] Specifically, these nanomaterials facilitate the development of innovative strategies for detecting disease biomarkers with increased sensitivity, specificity, and simplicity. For instance, fusion‐inspired nanomaterials have been employed for the detection of viral infections such as influenza, human immunodeficiency virus (HIV), and severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) by selectively binding to viral particles and amplifying detection signals.[

15

] In cancer diagnostics, they have enabled the non‐invasive detection of tumor‐derived exosomes carrying cancer‐specific biomarkers, offering considerable potential for early detection and real‐time monitoring of disease progression.[

17

]

Despite their potential, several challenges remain in translating fusion‐inspired nanomaterials into clinical applications. A key focus is to ensure the reproducibility and reliability of the detection platform through the optimization of membrane stability, fusion efficiency, and adaptability to complex biological environments.[

18

] Achieving a balance between the stability and fusogenic activity of these nanomaterials necessitates the careful design and optimization of lipid composition,[

19

] particle size,[

20

] and surface functionalization.[

21

] Addressing these important challenges is crucial for enhancing the clinical applicability of these nanosystems. Therefore, this review aims to provide a comprehensive overview of membrane fusion‐inspired nanomaterials, with a focus on their design principles, functionalization strategies, signal amplification approaches, control of fusion systems, and applications in infectious disease detection and cancer diagnostics (Figure

1
). By examining the latest advancements and challenges in this rapidly evolving field, we highlight the transformative potential of these biomimetic nanosystems to revolutionize diagnostic approaches and pave the way for more effective and personalized healthcare solutions.

2
Biological Mechanism of Membrane Fusion
Membrane fusion is a highly regulated and intricate process that involves the merging of two lipid bilayers into a single continuous membrane.[

22

] This process is orchestrated by an interplay of protein machinery and lipid dynamics, which work together to overcome the significant energetic barriers associated with membrane merging.[

23

] In biological systems, fusion occurs during vesicle trafficking,[

24

] organelle dynamics,[

25

] and viral entry into host cells (Figure

2
).[

23
,
26

] Central to these processes are fusion proteins and lipid intermediates that mediate the sequential stages of fusion, including initial membrane recognition, hemifusion stalk formation, hemifusion diaphragm expansion, pore opening, pore expansion, and complete fusion.[

26
,
27

] These intricate mechanisms serve as a foundational inspiration for designing membrane fusion‐inspired nanomaterials, which replicate these biological steps for use in synthetic systems (Figure

3
).
Understanding the molecular basis of membrane fusion requires a detailed examination of its key components. Proteins such as SNAREs in intracellular fusion[

24

] and viral fusion proteins such as hemagglutinin (HA) and glycoprotein 41 (gp41) in viral entry[

4

] play pivotal roles by inducing conformational changes that drive membrane proximity and bilayer destabilization. Lipids also contribute by forming highly curved intermediates, such as stalk and hemifusion states, that facilitate the merging of bilayers.[

5

] These interactions not only ensure efficient fusion in biological systems but also provide a blueprint for replicating these dynamics in biosensing and diagnostic applications, where precision and efficiency are critical.
This section explores the biological mechanisms underlying membrane fusion, focusing on intracellular processes such as vesicle trafficking and SNARE‐mediated fusion, as well as viral entry mechanisms involving specialized fusion proteins. By detailing these natural systems, we can establish a foundation for the design of fusion‐inspired nanomaterials tailored for advanced biosensing and diagnostic applications.
2.1
Intracellular Membrane Fusion
2.1.1
Vesicle Trafficking, Organelle Fusion, and Cellular Transport
Vesicle trafficking, organelle fusion, and cellular transport are all vital processes that help to maintain organelle specificity, ensuring efficient cargo transfer between organelles and preserving overall cellular function and homeostasis.[

24

] In eukaryotic cells, these mechanisms are pivotal for protein secretion, metabolic control, and stress responses. Vesicle trafficking selectively transports cargo across organelles, thereby supporting vital functions such as protein secretion, the recycling of membrane components, and the maintenance of organelle integrity. This process is critical for cellular homeostasis, as it regulates protein transport and retrieval. Disruption in vesicle trafficking can result in improper cargo delivery, impaired organelle function, and widespread cellular dysfunction, contributing to metabolic imbalances, accumulation of misfolded proteins, and increased risks of diseases such as cardiovascular and neurodegenerative conditions.
Organelle fusion entails the merging of two discrete organelles, allowing the exchange of their respective contents. This process is crucial for signaling, metabolism, organelle maintenance, and other cellular functions. For example, mitochondrial fusion supports mitochondrial health by mixing the contents of individual mitochondria, reducing damage, and stabilizing genetic material.[

28

] Mitofusins (Mfn1 and Mfn2) are key proteins for outer mitochondrial membrane fusion, while optic atrophy 1 is crucial for inner mitochondrial membrane fusion.[

28
,
29

] Similarly, SNARE proteins mediate the fusion of various organelles by facilitating membrane merging.[

24

]

The mechanism of organelle fusion progresses through five key stages. Initially, organelles are brought into proximity by tethering proteins, which facilitate membrane alignment.[

30

] Subsequently, the membranes accumulate at the contact site, priming them for fusion. In the hemifusion stage, the outer leaflets of the membranes merge, forming a hemifusion stalk, while the inner leaflets remain distinct.[

29
,
30

] This is followed by the complete fusion stage, where the inner leaflets merge, leading to full integration of the organelles and mixing of their contents. Once fully fused, the organelles share their internal components, enabling interaction and coordination in cellular functions.[

28

] Notable instances of organelle fusions include mitochondrial fusion, endoplasmic reticulum (ER) membrane fusion,[

31

] vesicular transport,[

24

] and peroxisome fusion.[

29
,
30
,
31

] Each of these processes has been determined to play a distinct role in the maintenance of cellular health and adaptability. For instance, ER membrane fusion is vital for lipid and protein synthesis, while peroxisome supports lipid metabolism, detoxification, and protein processing.[

28
,
31

]

Insights into vesicle trafficking and organelle fusion mechanisms have significant implications for targeted and controlled drug delivery, as well as precise diagnostics. Through the emulation of these natural processes, researchers can engineer drug carriers that deliver therapeutic agents with enhanced precision to target cells or tissues, optimizing treatment efficacy while minimizing off‐target effects. Furthermore, these principles can be applied to develop advanced diagnostic tools that exploit the specificity and efficiency of natural fusion events to detect disease biomarkers with high sensitivity.

2.1.2
SNARE Protein Complexes and Their Roles in Intracellular Fusion
SNARE proteins are fundamental to intracellular membrane fusion, facilitating the specific and targeted merging of lipid bilayers. A prominent example is the SNARE‐mediated fusion of synaptic vesicles with the plasma membrane at neuronal synapses, an essential process for neurotransmitter release. In this mechanism, a vesicular SNARE (v‐SNARE) pairs with a target membrane SNARE (t‐SNARE) to form a stable SNARE complex.[

32

] This complex subsequently drives the fusion of the vesicle membrane with the presynaptic membrane, facilitating the release of neurotransmitters into the synaptic cleft.[

32
,
33

] The formation of the SNARE complex overcomes the energetic barriers to fusion by establishing a highly stable four‐helix bundle, generating the mechanical force required to merge the membranes.[

32
,
34

] SNARE proteins function in coordination with regulatory proteins, including N‐ethylmaleimide‐sensitive factor (NSF) and soluble NSF attachment proteins, which mediate their assembly and disassembly. This regulatory system allows for the recycling of SNARE proteins, enabling repeated rounds of fusion, which are critical for processes such as neurotransmitter release.[

35

] The specificity of SNARE proteins is further enhanced by the presence of distinct SNARE proteins on specific organelles and vesicles, ensuring that vesicles fuse exclusively with their target membranes. This compartmental specificity is crucial for maintaining cellular organization and functional integrity.[

32a,b

]

Lipid interactions also play an important role in stabilizing the membranes and facilitating the docking and assembly of SNARE complexes. This proximity is essential for the formation of the fusion pore, which represents the final stage of membrane merging.[

32a,b

] During the hemifusion stage, lipid interactions enable the mixing of the outer leaflets of the bilayers without complete fusion.[

36

] This intermediate state minimizes leakage or cargo loss while preparing the membranes for full fusion. The specific lipid composition of membranes has a significant effect on fusion efficiency. For example, lipids such as phosphatidylserine (PS) or lysolipids can induce membrane curvature, promoting close apposition of the lipid bilayers and reducing the energy barrier to fusion.[

37

] These lipids help to create a favorable environment for SNARE‐mediated membrane fusion. The mechanisms of SNARE‐mediated fusion provide deep insights into cellular communication and organization. These principles can inspire the design of synthetic systems for biomedical applications, where controlled and well‐defined membrane fusion is critical, including targeted drug delivery and diagnostic tools.

2.2
Virus Membrane Fusion with Host Cells
2.2.1
Viral Entry Mechanism via Membrane Fusion
Viruses adopt membrane fusion at various stages during infection, leveraging their structural components to interact with host cells. Central to this mechanism are viral envelope glycoproteins, which mediate recognition and binding to specific receptors on the host cell surface and initiate a series of molecular events for membrane fusion.[

38

] This receptor‐binding event determines viral specificity and host cell tropism, dictating the range of cell types a virus can infect.[

39

] For example, the influenza virus binds to sialic acid residues on epithelial cells,[

40

] while the HIV targets cluster of differentiation 4 (CD4) and chemokine co‐receptors on immune cells.[

41

]

Following receptor engagement, membrane fusion proceeds through distinct pathways depending on the virus. Fusion occurs directly at the plasma membrane in some cases, such as paramyxoviruses.[

18a

] In contrast, many other viruses (e.g., influenza and SARS‐CoV‐2) undergo internalization via endocytosis, with fusion triggered in intracellular compartments, such as endosomes or lysosomes.[

38a

] These compartments provide environmental cues, including acidic pH or the presence of host proteases, that activate fusion proteins embedded in the viral envelope.[

42

] For instance, acidification within endosomes can induce conformational changes in viral glycoproteins, destabilizing both the viral and host membranes and lowering the energy barrier for fusion.[

43

] Additionally, the composition of the host cell membrane, particularly the presence of cholesterol and sphingolipids, supports membrane curvature and microdomain formation, further facilitating the fusion events.[

44

] These factors highlight the interplay between viral structural components and host membrane properties, underscoring the complexity of viral fusion.
Membrane fusion is pivotal for viral entry, allowing the nucleocapsid containing the viral genome to be delivered into the host cytoplasm. Once inside, the viral genome is uncoated, initiating the replication cycle.[

45

] A failure at the fusion stage effectively aborts the infection, making it a prime target for antiviral strategies.[

46

] For example, fusion inhibitors, such as enfuvirtide for HIV, disrupt this critical step, preventing the establishment of infection.[

47

] The timing and localization of fusion also influence viral pathogenesis. Viruses like Ebola fuse within late endosomal compartments, ensuring that their genomes are released near host replication machinery.[

48

] Such spatial and temporal control over fusion enhances viral survival and propagation within the host.
Insights into viral fusion mechanisms have inspired the development of nanomaterials engineered to mimic these processes. Researchers have created nanoparticles that incorporate viral fusion peptides or proteins, which can trigger membrane fusion upon interaction with target cell membranes.[

12
,
49

] These fusion‐inspired nanomaterials can be fine‐tuned to selectively target specific cells or tissues, making them ideal candidates for diagnostic applications.[

50

] Beyond diagnostics, the potential for therapeutic and imaging applications is significant, given the ability to manipulate viral fusion mechanisms in nanomaterials.[

51

] Fusion‐inspired nanomaterials can be integrated with molecular imaging techniques to detect viral particles or infected cells with high specificity. Additionally, these materials can serve as platforms for drug delivery systems, where fusion‐triggered release of therapeutic agents can be precisely controlled. By studying viral fusion, researchers can not only gain a deeper understanding of viral pathogenesis but also unlock new opportunities for innovative diagnostic tools and therapeutic strategies.

2.2.2
The Roles of Fusion Proteins
Fusion proteins are specialized viral surface proteins that mediate the fusion of the viral envelope with the host cell membrane.[

52

] These proteins undergo conformational changes triggered by external signals such as receptor binding, pH alterations, or the presence of ions. This results in the destabilization of membranes and the initiation of the fusion process. Notable examples include HA in influenza, gp41 in HIV, and the spike protein in SARS‐CoV‐2, which serve as critical models for understanding the molecular mechanisms of membrane fusion. A well‐studied example is the influenza virus, where the HA protein binds to sialic acid receptors on the host cell surface. Upon endocytosis, the acidic environment inside the host cell induces a conformational change in the HA protein, exposing a hydrophobic fusion peptide that integrates into the host membrane.[

53

] This action drives the viral and host membranes together, enabling the release of the viral genome into the host cell. A similar process occurs in the case of HIV, where the envelope protein gp120 binds to CD4 receptors on T‐cells, followed by interaction with the chemokine receptor CCR5 or CXCR4.[

54

] This binding instigates a conformational change in the viral gp41, resulting in the formation of a six‐helix bundle that facilitates membrane fusion. This fusion process involves the insertion of hydrophobic regions of the fusion protein into the host membrane, creating an intermediate fusion pore that ultimately allows the viral genome to enter the host cell.
The function of fusion proteins extends beyond the mere facilitation of membrane merging; they also interact with lipids present in both the viral envelope and the host cell membrane, generating curvature and destabilizing the lipid bilayer. This interaction gives rise to fusion intermediates, such as the hemifusion state, which bring membranes into proximity, lowering the energy barrier for complete fusion. The molecular choreography between protein and lipid dynamics ensures the efficient delivery of viral material into the host cell cytoplasm.
It is therefore important to understand the molecular mechanisms of fusion proteins for the design of fusion‐inspired nanomaterials. The incorporation of viral fusion proteins or peptides into nanomaterials enables researchers to mimic natural membrane fusion processes, offering promising applications in diagnostics, targeted drug delivery, and therapeutic interventions. For instance, fusion proteins can be adapted to enhance the targeting specificity of diagnostic nanomaterials by mimicking viral entry pathways, enabling these systems to selectively interact with infected cells or viral particles.[

55

] This capability can not only improve the sensitivity and accuracy of viral detection but also enhance the precision of therapeutic delivery, offering significant advantages in the fight against viral diseases.
In summary, while the processes of intracellular and viral membrane fusion share common mechanistic steps, including initial membrane recognition, hemifusion stalk/diaphragm formation, pore formation and expansion, and complete fusion, they differ significantly in their molecular regulators, initiation triggers, and biological contexts. Both processes rely on highly curved lipid intermediates and precise fusion protein‐lipid interactions to overcome the energetic barriers of membrane merging. However, intracellular fusion, exemplified by vesicle trafficking and organelle dynamics, is driven by SNARE protein complexes and accessory regulators with strict compartmental specificity. Conversely, viral membrane fusion is typically mediated by viral envelope glycoproteins responding to external stimuli, such as receptor binding or pH shifts, which enables membrane fusion at the plasma membrane or within endosomal compartments. These distinctions influence the design of membrane fusion‐inspired nanomaterials. SNARE‐like intracellular systems provide a blueprint for constructing nanocarriers that deliver cargo effectively within the cytoplasm, while viral fusion mechanisms inspire the creation of stimuli‐responsive nanomaterials that mimic virus‐host interactions for precise targeting and diagnostic activation. The integration of insights from both intracellular and viral fusion models enables the rational engineering of synthetic systems with enhanced functional control, selectivity, and applicability across diverse biomedical environments.

3
Design Principles of Fusion‐Inspired Nanomaterials
Bio‐inspired or biomimetic nanomaterials replicate key structural and functional features of natural systems, driving advancements in healthcare applications such as targeted drug delivery, diagnostics, and biomarker detection. They are particularly well‐suited for mimicking membrane fusion pathways, which are integral to molecular transport and viral infection in vivo. By emulating the physicochemical properties of cellular and viral membranes, they offer precise control over fusion events for targeted interactions with biological systems. The design of these nanomaterials necessitates careful consideration of factors, including nanoparticle type, composition, size, surface charge, and membrane properties. Functionalization with ligands, antibodies, peptides, or DNA aptamers further refines their specificity and responsiveness to stimuli such as pH or ion concentration changes (Figure

4
).
Liposomes, spherical vesicles composed of lipid bilayers, exemplify this design approach. They are among the most commonly used nanomaterials in replicating cell or viral membranes due to their structural similarity and versatility.[

56

] The lipid composition exerts a pivotal influence on membrane properties (i.e., fluidity and curvature) and regulates fusion dynamics.[

57

] For instance, the inclusion of phospholipids, glycolipids, and/or cholesterol allows fine‐tuning of the stability and fusion efficiency of the bilayers.[

58

] Additionally, exosome‐mimicking nanoparticles, which combine lipid bilayers with proteins, replicate the surface features of exosomes, allowing them to interact efficiently with target membranes.[

59

] Incorporating polyethylene glycol (PEG) into lipid bilayers provides additional functional advantages, such as reducing immune recognition and prolonging circulation time, making them particularly beneficial for diagnostic applications.[

59b

] Such functionalization improves selectivity and enables nanoparticles to evade non‐specific interactions to reach their intended target.[

60

] By carefully manipulating these lipid components, researchers can design nanomaterials capable of initiating fusion under precise biological conditions. In light of these considerations, this section will explore the design principles of lipid‐based nanomaterials, focusing on their ability to replicate the complexities of the natural fusion processes while optimizing their functionality for advanced biomedical applications.
3.1
Structural Components and Functionalization
3.1.1
Lipid Compositions and Types of Nanomaterials
The lipid composition of nanomaterials is a fundamental aspect in the design of systems that replicate the intricacies of natural membrane fusion. The utilization of lipids such as phospholipids, sphingolipids, and glycolipids is imperative in the emulation of the structural and functional characteristics inherent to biological membranes. Liposomes, lipid‐based nanomaterials, exemplify this approach by forming bilayer structures that closely resemble cellular membranes. These spherical vesicles are highly versatile and capable of encapsulating both hydrophilic and hydrophobic substances, making them suitable for various biomedical applications, including diagnostics and drug delivery. For example, phosphatidylcholine (PC) provides stability and flexibility to bilayers, ensuring compatibility with cellular targets, while phosphatidylethanolamine (PE) promotes negative membrane curvature, which is crucial for destabilization and fusion.[

61

] The interplay between the bilayer stability provided by PC and the fusogenic activity of PE facilitates processes like hemifusion and lowers the energy barrier for membrane fusion, advancing applications in targeted drug delivery, gene therapy, and vaccine development.
Sphingolipids and glycolipids enhance fusogenic systems by enabling specific binding and structural rigidity, supporting precise interactions with specialized cell types, such as neural cells enriched in gangliosides or epithelial cells expressing glycosphingolipids.[

62

] The specificity of these interactions is attributable to the presence of unique carbohydrate moieties or ceramide backbones that interact selectively with cellular receptors or lectins. For example, sialic acid‐containing glycolipids, such as GM1 ganglioside, have been shown to play critical roles in receptor clustering and signal transduction.[

63

] Additionally, the structural rigidity of sphingolipids stabilizes lipid bilayers, prevents premature degradation, and enhances nanoparticle stability in complex environments.[

64

]

Cholesterol fulfills a dual role in enhancing membrane fluidity and stability, optimizing nanomaterial performance during circulation and fusion. The addition of 30–50 mol% cholesterol to lipid bilayers in liposomal drug carriers has been shown to reduce membrane permeability, preventing premature drug leakage and maintaining the flexibility required for membrane fusion.[

65

] Cholesterol achieves this by intercalating between phospholipids, which further modulates packing density and phase transitions and stabilizes the bilayer in the liquid phase.
On the other hand, the regulatory effect of cholesterol on membrane fluidity can vary depending on the lipid environment.[

19b

] In membranes rich in saturated lipids like sphingomyelin or dipalmitoylphosphatidylcholine (DPPC), cholesterol intercalates between the tightly packed acyl chains, disrupting the gel‐like, highly ordered arrangement. This transition forms a liquid‐ordered phase, which retains structural stability while enhancing flexibility. For instance, cholesterol has been demonstrated to impede the gel‐phase transition of DPPC, ensuring fluidity even at low temperatures, a property critical for sustaining membrane dynamics.[

66

] Conversely, in unsaturated lipid environments, such as those containing dioleoylphosphatidylcholine (DOPC), cholesterol occupies the spaces generated by the kinks in unsaturated lipid tails. This interaction reduces excessive fluidity and stabilizes the membrane, making it suitable for drug delivery applications.[

67

] The dual effect of cholesterol—enhancing fluidity in saturated lipid environments and reducing fluidity in unsaturated lipid environments—optimizes membrane behavior for dynamic processes such as fusion. For example, in viral fusion, cholesterol‐rich membranes promote hemifusion and fusion pore formation by stabilizing negative curvature in the lipid bilayer.[

23

] These findings highlight the critical role of cholesterol in fine‐tuning the membrane properties of nanomaterials to support dynamic fusion processes.
The incorporation of functional lipids, such as PEG‐tethered or pH‐sensitive lipids, further enhances the versatility of these nanomaterials for biomedical applications. PEGylation, the attachment of PEG to a nanomaterial, has been shown to extend circulation time by forming a hydrophilic barrier that reduces non‐specific interactions with proteins and immune cells.[

68

] This prolonged circulation is vital for sensitive disease detection, increasing the likelihood of encountering target biomarkers or pathogens. However, the effects of PEGylation are highly dependent on the PEG chain length.[

69

] In this study, antibody‐loaded poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles modified with long PEG chains (PEG5k) showed superior mucus penetration and colloidal stability in gastrointestinal environments but also acquired near‐neutral surface charge and increased hydrophilicity. These properties led to reduced affinity for positively charged inflamed tissues and diminished therapeutic efficacy. In contrast, PLGA nanoparticles coated with shorter PEG chains (PEG2k) retained a more negative surface charge, enabling stronger electrostatic interactions with target sites and enhanced local accumulation. PEGylation can also interfere with direct interactions between PEG lipid‐based nanocarriers and cell membranes, thereby reducing absorption rates and potentially impairing the membrane fusion capability. This phenomenon, referred to as the PEG dilemma, has been observed in stealth liposomes and arises when the PEG layer compromises fusion efficiency at the intermediate surface layer.[

70

] These results highlight the need to optimize PEG content to achieve a balance between extended circulation and site‐specific targeting while minimizing unintended trade‐offs in membrane fusion performance.
In addition, pH‐sensitive lipids, such as dioleoylphosphatidylethanolamine (DOPE) combined with cholesteryl hemisuccinate (CHEMS), offer unique advantages for controlled cargo release. These lipids form stable bilayers at neutral pH but undergo structural changes in acidic conditions.[

71

] Under acidic conditions, the protonation of CHEMS destabilizes the bilayer and induces a transition to a hexagonal phase, promoting membrane fusion and targeted cargo release. This approach is particularly effective in tumor therapy, where the acidic tumor microenvironments activate the system, minimizing off‐target effects and enhancing therapeutic specificity.
Beyond PEGylation and pH responsiveness, recent investigations by Choi et al. have revealed that the modification of nanomaterials with alkyl functional groups serves as a highly effective means of directing the fate of nanoparticles within and between cells. Their work demonstrated that the introduction of low percentages of linear long‐chain alkyl groups (e.g., dodecyl or octadecyl) into the PEG shell of gold nanoparticles significantly increased cellular uptake by up to 10‐ to 100‐fold, depending on the cell type.[

72

] This uptake was shown to occur predominantly via filopodia‐mediated mechanisms and resulted in substantial cytoplasmic accumulation, highlighting a route distinct from classical endocytosis pathways. In addition to internalization, the structure of the alkyl group significantly influenced nanoparticle exocytosis.[

73

] Cyclododecyl‐modified particles, for instance, were efficiently expelled from phagocytic cells such as macrophages but retained within epithelial cancer cells, whereas branched and aromatic groups modulated release kinetics in cell‐type‐specific ways. Furthermore, these alkyl‐modified gold nanoparticles were found to engage with unconventional exocytic pathways, including packaging into extracellular vesicles (EVs).[

72

] Notably, dodecyl‐functionalized gold nanoparticles induced EV production in keratinocytes and altered the EV composition and function, offering new opportunities for designing EV‐based delivery systems and diagnostics. Altogether, these findings establish alkyl group engineering as a versatile and powerful molecular strategy to tailor nanoparticle‐cell interactions, intracellular trafficking, and intercellular communication.

3.1.2
Particle Size and Surface Charge

Particle Size: The size of nanoparticles is a crucial parameter in the design of fusion‐inspired nanomaterials, as it dictates their interaction with target membranes and overall functional efficacy. Nanoparticles with a size range of 10–200 nm are particularly advantageous for applications requiring cellular uptake and membrane fusion. This size range provides a high surface area‐to‐volume ratio, which facilitates close interaction with cellular or viral membranes and enhances fusion efficiency.[

74

] Theoretical models support this optimization. For instance, stalk formation energy can decrease by up to 31 kBT for 20 nm vesicles compared to flat membranes, while fusion pore formation energy barriers drop by ≈17 kBT in highly curved vesicle–vesicle configurations. This reduction in energy barriers significantly increases the likelihood and efficiency of membrane fusion. In practical applications such as small interfering RNA (siRNA) delivery, nanoparticles smaller than 200 nm achieved gene silencing efficiencies of ≈72%–90%, emphasizing the need to maintain nanoscale dimensions for therapeutic effectiveness.[

75

] However, nanoparticles smaller than 10 nm, such as ultrasmall gold nanoparticles (AuNPs), face limitations due to rapid systemic clearance, with the reported renal elimination rate as high as 46.71%, primarily driven by renal filtration and uptake by the reticuloendothelial system. This short circulation time constrains their therapeutic window.[

76

] Nonetheless, the tunability of nanoparticle size offers a significant design advantage, enabling targeted delivery to specific cells or tissues. For example, studies have shown that lipid nanoparticles with diameters ranging from 76.5 to 117 nm preferentially target hepatocytes in the liver, while increasing the particle size up to 200 nm shifts the selectivity toward liver sinusoidal endothelial cells.[

77

]

In contrast, giant unilamellar vesicles (GUVs), which range from 10 to 100 µm in size, offer distinct advantages for ex vivo diagnostics and mechanistic studies.[

78

] Their large size mimics the structural complexity of biological cells, making them valuable for advanced diagnostic platforms. Furthermore, GUVs can encapsulate larger quantities of diagnostic reagents, thereby amplifying detection signals. Their extensive surface area also supports the incorporation of functional biomolecules, such as ligands, antibodies, or receptors, increasing their sensitivity and selectivity in detecting biomarkers or pathogens.[

79

] Their visibility under standard optical microscopy enables real‐time observation of fusion events and molecular interactions. However, due to their inability to penetrate biological barriers or circulate effectively in vivo, GUVs are limited to diagnostic systems that do not require systemic delivery. Therefore, tailoring nanoparticle size to the intended application–smaller nanoparticles for systemic delivery and larger ones for ex vivo diagnostics–maximizes functionality and efficiency in specific biomedical contexts.

Surface Charge: Surface charge is another important parameter that regulates nanoparticle‐membrane interactions. Positively charged liposomes exhibit strong electrostatic attraction to negatively charged liposomes composed of PS and phosphatidylinositol (PI), which are abundant on the inner leaflet of the plasma membrane.[

80

] This attraction promotes membrane proximity and fusion events, increasing the likelihood of successful cargo delivery. Conversely, negatively charged nanoparticles, while less fusogenic due to electrostatic repulsion, offer enhanced colloidal stability and reduced aggregation, which are critical for designing reliable and stable diagnostic systems. Furthermore, the mimicry of natural membrane charge properties by negatively charged nanoparticles enables compatibility with diverse targets, including cells, exosomes, bacteria, and viruses. For instance, the negative charge of nanoparticles facilitates efficient interaction with exosomes, which are enriched with PS on their surfaces, rendering them effective for uptake and signaling applications.[

81

] Similarly, bacterial membranes composed of anionic lipids (e.g., cardiolipin and phosphatidylglycerol) align with the properties of negatively charged nanoparticles, enhancing their potential in antimicrobial or bacterial diagnostic systems.[

82

] In the context of viruses, negatively charged nanoparticles can mimic viral lipid envelopes, improving their interaction with host cells for targeted delivery or diagnostic purposes.[

83

]

3.1.3
Membrane Fluidity and Curvature

Membrane Fluidity: Membrane fluidity is a critical determinant of fusion efficiency, governing the lateral movement of lipids within the bilayer and enabling dynamic rearrangements that are essential for membrane deformation.[

84

] High fluidity facilitates the formation of transitional fusion structures, such as hemifusion states and stalk intermediates, which are vital for merging two lipid bilayers. Unsaturated lipids containing one or more double bonds in the fatty acid chain, such as DOPC, linoleic acid‐based phospholipids, docosahexaenoic acid‐based lipids, significantly enhance membrane fluidity by introducing kinks in the chains, preventing tight packing, and increasing flexibility.[

85

]

Cholesterol also plays a key role in modulating membrane fluidity.[

86

] In saturated lipid membranes (e.g., DPPC), cholesterol intercalates between tightly packed fatty acid chains, reducing rigidity and forming a liquid‐ordered phase. In contrast, in unsaturated lipid membranes (e.g., DOPC), cholesterol fills gaps created by the kinks in the lipid tails, reducing excessive fluidity and stabilizing the bilayer. For example, liposomes with optimal cholesterol content (30–50 mol%) have been shown to improve bilayer stability by modulating the membrane fluidity while maintaining sufficient flexibility for dynamic interactions with cellular membranes.[

87

] Similarly, modified sterols, such as ergosterol found in fungal membranes, have been incorporated into liposomal formulations to modulate membrane dynamics, improve stability, and control drug release.[

88

]

Liposomes with high membrane fluidity, composed of unsaturated lipids (e.g., DOPC, 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine), have demonstrated superior fusion and cellular uptake compared to rigid formulations, such as DPPC‐based liposomes.[

89

] However, excessive fluidity, typically observed in liposomes composed exclusively of unsaturated lipids, can lead to structural instability.[

90

] This instability manifests as cargo leakage and reduced circulation time.[

91

] Incorporating stabilizing agents (e.g., cholesterol or PEG) or a blend of saturated and unsaturated lipids ensures a balance between fluidity and structural integrity, maximizing delivery efficiency while maintaining cargo stability.
The modulation of membrane fluidity has been validated using advanced analytical techniques, offering critical insights into how lipids and sterols influence bilayer dynamics. For example, fluorescence recovery after photobleaching (FRAP) has been employed to measure the lateral diffusion rates of lipids, a key indicator of membrane fluidity.[

92

] FRAP analysis showed that increasing cholesterol concentration in DOPC‐rich membranes reduces diffusion rates, demonstrating the role of cholesterol in decreasing fluidity. Similarly, electron spin resonance spectroscopy, which monitors the behavior of spin‐labeled lipids, has revealed that cholesterol increases the order of saturated lipid bilayers while decreasing the disorder of unsaturated ones.[

93

] Furthermore, differential scanning calorimetry has been used to investigate phase transition temperatures, showing that cholesterol broadens and shifts the transition phase of DPPC membranes, confirming its role in fluidity modulation.[

71

]

Membrane Curvature: Membrane curvature is equally pivotal, as it influences the ease of bilayer deformation and fusion.[

94

] High curvature generates stress within the bilayer, destabilizing the membrane and lowering the energy barrier for intermediate formation. Nanomaterials with high curvature are carefully engineered using specific lipid compositions that promote the formation of non‐lamellar phases, facilitating interactions with target membranes, such as those of viruses or cancer cells. For example, cone‐shaped lipids like DOPE induce negative curvature due to their small headgroup and bulky hydrophobic tails, which promote adhesion to the membrane and increase the fusion probability.[

57b

] At concentrations of 50–70 mol%, DOPE transforms the lipid bilayer into an inverted hexagonal phase, a structure conducive to membrane fusion, while mixing of unsaturated fatty acids (10–20 mol%) can induce packing defects and further amplify the membrane curvature.[

95

]

High‐curvature nanoparticles (50–100 nm) are particularly effective in penetrating tight cellular junctions, enabling precise targeting of biomarkers in these localized environments.[

15
,
96

] Surface modifications with short‐chain PEG or targeting ligands can introduce asymmetry and curvature, potentially enhancing membrane interactions and promoting processes such as cellular uptake and endosomal escape.[

68

] Thermal treatments, including controlled heating and rapid cooling, can further modulate curvature by inducing phase transitions.[

97

] Conversely, low‐curvature membranes are more stable and less prone to spontaneous fusion, making them ideal for applications requiring prolonged cargo delivery. Nanoparticles with low curvatures, such as large unilamellar vesicles composed of saturated lipids like DPPC and cholesterol, provide rigidity and reduced permeability, ensuring sustained drug release.[

98

] For instance, DPPC/cholesterol liposomes have been used to encapsulate doxorubicin, demonstrating extended circulation times and sustained drug release in cancer therapy.[

99

] Multi‐lamellar vesicles, which consist of DPPC, cholesterol, and phosphatidylglycerol, offer additional barriers to drug leakage, making them practical for depot formulations such as leuprolide acetate for hormone therapy.[

100

] Solid lipid nanoparticles and nanostructured lipid carriers combine low‐curvature solid lipids, such as glyceryl monostearate, with liquid lipids like oleic acid to fine‐tune release kinetics.[

101

] These systems achieve sustained release profiles while maintaining structural integrity, as demonstrated in preclinical studies with paclitaxel.[

102

]

In the field of diagnostics, controlled curvature allows for precision in the detection of biomarkers by influencing the likelihood of interactions with specific target cells or pathogens. High‐curvature nanoparticles improve the presentation and accessibility of surface ligands, reducing steric hindrance and increasing the probability of binding to densely packed or nanoscale biomarkers. For example, nanoparticles with high curvature have been shown to improve the detection of HIV‐1 envelope glycoprotein gp120 by mimicking the nanoscale topography of viral surfaces.[

103

] Similarly, high‐curvature liposomes have been used to target the curved membranes of exosomes, enhancing detection specificity for exosomal proteins.[

103

] In contrast, low‐curvature nanoparticles excel in long‐term biomarker detection, as their stability prevents premature aggregation and ensures consistent interactions in the bloodstream.[

104

]

The modulation of membrane curvature has been validated using various advanced analytical techniques. Small‐angle X‐ray scattering (SAXS) detects structural phases in lipid systems.[

105

] In SAXS, high‐curvature materials exhibit characteristic scattering patterns indicative of non‐lamellar phases, confirming the desired membrane architecture.[

106

] Cryo‐electron microscopy provides direct visualization of tightly curved regions in liposomes or nanoparticles.[

107

] Dynamic light scattering measures the hydrodynamic diameter and polydispersity of nanoparticles, with smaller diameters (50–100 nm) correlating to higher curvature.[

108

] Förster resonance energy transfer (FRET)‐based fusion assays confirm enhanced fusion efficiency in high‐curvature liposomes,[

109

] while atomic force microscopy and surface plasmon resonance provide insights into curvature‐specific interaction dynamics and binding kinetics.[

110

] Additionally, in vitro dissolution studies reveal that low‐curvature nanoparticles typically exhibit slower and more sustained drug release compared to high‐curvature counterparts.[

111

]

By carefully controlling curvature through lipid composition, environmental triggers, and nanoparticle design, researchers can create tailored nanomaterials optimized for their intended applications. This approach is particularly valuable in diagnostics, where precision in fusion and interaction dynamics significantly improves sensitivity and selectivity. Furthermore, integrating curvature control into nanoparticle design bridges the gap between natural membrane fusion processes and practical biomedical applications.

3.2
Functionalization for Fusion Control
3.2.1
Functionalization with Viral Fusion Proteins
Surface functionalization is critical to designing fusion‐inspired nanomaterials, enabling specific and controllable interactions that drive efficient membrane fusion. This process facilitates targeted applications such as biomolecule detection, drug delivery, and diagnostic platforms by enhancing the selectivity and efficiency of fusion events. Functionalization strategies utilize viral fusion proteins and SNARE‐mimicking ligands to replicate natural membrane fusion mechanisms or to engineer programmable fusion processes, providing advanced tools for addressing challenges in biosensing and therapeutics.
Viral fusion proteins, such as HA, neuraminidase (NA), and gp120, are widely adopted for functionalizing liposomes to induce controlled membrane fusion. These proteins mimic natural viral entry pathways, involving conformational changes triggered by specific stimuli, such as intracellular pH shifts or protease activity. Upon activation, these proteins expose their fusion‐mediating subunits, which insert into lipid bilayers, destabilize the membranes, and promote fusion. For example, HA undergoes structural rearrangements in acidic environments, resulting in membrane merging and cargo exchange. This mechanism has been adapted for detecting breast cancer‐associated exosomal microRNAs (miRNAs), where HA‐functionalized liposomes replicate the fusion process of the influenza virus to achieve rapid and accurate classification of cancer from healthy samples.[

112

]

To incorporate viral fusion proteins into liposome membranes, two main strategies have been employed. One approach involves the use of surfactants, such as N‐octyl‐β‐D‐glucopyranoside (OG), to integrate exogenous fusion proteins. These surfactants temporarily disrupt the lipid bilayer, facilitating the insertion of hydrophobic domains of the fusion proteins while preventing protein aggregation. For example, Park et al. used OG to reconstitute HA proteins into lipid bilayers, developing nanosensors for exosomes by resembling sialic acid‐HA binding and HA‐mediated membrane fusion.[

112a

] This process requires precise surfactant concentration and rigorous washing steps to preserve protein functionality. Alternatively, cell‐derived membranes embedded with transiently expressed fusion proteins can be isolated and reconstituted into liposomes. Gao et al. employed this method using cell membranes containing the HA‐NA protein and fusion protein of the human parainfluenza virus to preserve the native functionality of proteins.[

112b

] These strategies ensure the integration of active fusion proteins into lipid membranes, enabling efficient and specific membrane fusion.

3.2.2
Functionalization with SNARE‐Mimicking Ligands
SNARE‐mimicking ligands, including coiled coil‐forming peptides and DNA zippers, offer innovative solutions for inducing and regulating membrane fusion.[

113

] These ligands replicate the structural and functional behavior of the SNARE protein complex, which mediates natural vesicle fusion through a coiled‐coil motif formed by four helices. Coiled‐coil peptides, such as JR2EC/JR2KC or E/K pairs, dock vesicles by forming stable heterodimers, reducing the energy barrier for fusion.[

114

] The fusion process typically progresses through docking, semi‐fusion, and complete fusion, allowing controlled content mixing. Cholesterol‐tagged or lipid‐modified peptides are used to attach these ligands to liposomes, ensuring stable and efficient integration. Spacer molecules like ethylene glycol are incorporated to optimize steric accessibility and enhance hybridization efficiency.
DNA zippers, a pair of complementary DNA strands, represent another versatile approach for functionalization, leveraging the programmability and sequence‐specific hybridization properties of DNA. Complementary DNA strands anchored to liposomal membranes via lipid or cholesterol modifications hybridize to form a zipper‐like structure, bringing the membranes into close contact and destabilizing the bilayers to induce fusion. Unlike peptides, DNA‐mediated membrane fusion systems enable precise control over fusion events, as DNA can be easily modified to alter sequences, secondary structures, and hybridization methods. For example, Jumeaux et al. designed a hairpin DNA structure to block hybridization by binding to the toehold region of DNA zippers, which prevents liposome docking until activated by a target molecule.[

113b

] Upon target presence, the hairpin DNA undergoes a conformational change, initiating DNA hybridization and zipping, which triggers membrane fusion and content mixing. This generates a FRET signal, enabling highly sensitive detection of miRNA biomarkers through specific liposome membrane fusion. DNA zipper‐mediated membrane fusion has also been used to non‐destructively load therapeutic siRNA into target cell‐derived exosomes, producing hybrid exosome vesicles for tissue‐specific siRNA delivery.[

113a

] Zipper DNA‐mediated membrane fusion can be used not only for drug loading but also as an efficient strategy for intracellular drug delivery.
Moreover, functional DNA molecules such as aptamers and DNAzymes expand the versatility of DNA‐mediated membrane fusion systems. The incorporation of external stimuli‐responsive motifs (e.g., light‐sensitive cleavage sequences) into DNA sequences can be introduced into the sequence. For example, Sun et al. reported intracellular protein delivery using DNA‐mediated membrane fusion between liposomes and cells and demonstrated that this strategy successfully delivers exogenous protein cargo to the cytoplasm, bypassing the endosomal pathway and modulating cell function.[

115

] In addition, Willner et al. functionalized the surface of liposomes with hairpin‐shaped locked strands that can be cut by DNA aptamers and infrared light stimulation.[

116

] In particular, by loading upconversion nanoparticles inside the liposomes, they induced DNA cleavage through ultraviolet emission by near‐infrared irradiation, caused zipper DNA‐mediated membrane fusion between cells and liposomes coated with complementary sequences to deliver drugs, and demonstrated near‐infrared‐driven spatiotemporal cytotoxicity.

4
Membrane Fusion‐Inspired Nanomaterials in Biosensing and Diagnostics
Signal generation is crucial for translating membrane fusion events into measurable outputs in biosensing and diagnostic applications. Membrane fusion‐induced nanomaterials must produce reliable signals corresponding to specific fusion events, which can be detected using optical, electrochemical, or imaging‐based instruments. The integration of isothermal nucleic acid amplification and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated (Cas)‐based diagnostic platforms into those nanomaterials offers a robust approach for generating and amplifying signals. Isothermal amplification methods, including enzyme‐free and enzyme‐assisted techniques, are advantageous in that they do not require thermal cycling, rendering them compatible with fusion‐triggered systems. Furthermore, CRISPR/Cas systems leverage programmable guide RNAs to target disease‐specific nucleic acid sequences, providing superior specificity. Coupling CRISPR/Cas systems with membrane fusion nanomaterials enables precise detection of nucleic acids released during fusion events, facilitating highly accurate diagnostics for cancer and viral infections.
External stimuli, such as pH, ions, enzymes, and light, add precision and versatility to these diagnostic platforms. pH‐sensitive nanomaterials respond to acidic environments, such as tumor microenvironments or inflamed tissues, to release encapsulated agents or activate signals. Similarly, ion‐activated systems leverage elevated ion concentrations to induce site‐specific fusion.[

117

] Enzyme‐triggered systems use disease‐specific enzymes, such as proteases, to regulate fusion events, while light‐activated systems provide spatial and temporal control through photosensitive molecules. These stimuli‐responsive mechanisms enhance the specificity and efficacy of membrane fusion‐inspired nanomaterials, paving the way for multifunctional diagnostic tools capable of precise signal amplification and controlled fusion processes in clinical applications. The following section will thus focus on the practical strategies for employing membrane fusion‐inspired nanomaterials, with a particular emphasis on their ability to enhance signal amplification and attain precise control over fusion processes for sensitive and selective biosensing and diagnostic applications.
4.1
Signal Amplification Strategies
4.1.1
Isothermal Nucleic Acid Amplification
Isothermal nucleic acid amplification systems, encompassing enzymatic and non‐enzymatic approaches, are of pivotal significance for the sensitive detection of target biomolecules present at low abundance. These systems eliminate the need for thermal cycling, offering streamlined and efficient amplification in a range of settings, including point‐of‐care diagnostics. Enzymatic strategies, such as loop‐mediated isothermal amplification, exponential amplification reaction, and recombinase polymerase amplification (RPA), utilize DNA‐processing enzymes like DNA polymerases, ligases, nicking endonucleases, and recombinases, producing 106–109‐fold signal increases with exceptional sensitivity.[

118

] In contrast, non‐enzymatic methods, including catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR), achieve amplification through toehold‐mediated strand displacement reactions, harnessing strand hybridization and catalytic mechanisms to yield detectable outputs.[

119

] These non‐enzymatic systems are cost‐effective, programmable, and operate under mild reaction conditions, making them ideal for integration into advanced diagnostic platforms.
In toehold‐mediated strand displacement, a short single‐stranded toehold region initiates the exchange of complementary strands, driving amplification through sequential hybridization.[

120

] CHA employs hairpin DNA structures that catalytically open and close upon interaction with a target molecule, triggering a chain reaction for signal amplification. Similarly, HCR relies on alternating hybridization events between hairpin DNA probes to produce long double‐stranded products, amplifying the detection signal. Encapsulating these systems in fusogenic liposomes enables highly sensitive detection of biomarkers, particularly present in exosomes.
Recent advancements have demonstrated innovative applications of isothermal amplification approaches for the highly selective and sensitive detection of exosomal miRNAs.[

113
,
121

] For example, Xia et al. introduced an RNA walker‐mediated signal amplification system for exosomal miRNA detection (Figure

5

a).[

122

] The RNA walker probe performed autonomous multi‐step walking upon hybridization to a target miRNA, leading to amplified signals for highly sensitive detection. The system also incorporated DNA logic circuits, including AND, OR, and NOT gates, which processed signals from exosomal surface proteins to ensure selective fusion with target exosomes. After selective membrane fusion, the walker probes performed cascade amplification, significantly improving both sensitivity and specificity for the detection of exosomal miRNAs. Similarly, Sun et al. have developed a CHA‐ and DNAzyme‐based amplification system encapsulated in cationic fusogenic liposomes for single exosome analysis (Figure 5b).[

113d

] The system uses a pair of hairpin probes that undergo a cascade amplification process driven by a target miRNA within the exosome. The CHA‐DNAzyme circuit provides repeated hybridization and cleavage cycles, generating a robust fluorescence signal with remarkable sensitivity for the detection of trace exosomal miRNAs. This approach combines high amplification efficiency with the ability to preserve exosome integrity, making it particularly effective for the analysis of exosome‐associated biomarkers. Additionally, Hu et al. developed cationic lipid‐polymer hybrid nanoparticles tethered onto a chip to capture circulating EVs without the need for isolation (Figure 5c).[

121

] By encapsulating a catalyzed hairpin DNA circuit within these nanoparticles, they achieved sensitive imaging and quantification of low‐expression glypican‐1 mRNA in serum EVs. This method enabled differentiation between patients with early‐ and late‐stage pancreatic cancer, as well as distinguishing them from healthy donors and patients with benign pancreatic diseases.
The integration of signal amplifiers into fusion‐inspired nanomaterials represents a significant advancement in biosensing and diagnostics. These systems enable the identification of low‐abundance biomarkers critical for early disease diagnosis, enhancing the accuracy, reliability, and clinical relevance of diagnostic results. Future research should focus on optimizing nucleic acid‐based probe design, improving encapsulation efficiency in nanomaterials, and ensuring robust performance in complex biological environments.

4.1.2
CRISPR‐Based Sensing System
CRSIPR‐Cas systems are increasingly being utilized in diagnostic applications due to their exceptional ability to target and cleave nucleic acids with high specificity, thereby significantly improving the sensitivity and specificity of biosensing platforms.[

123

] These systems leverage the unique properties of CRISPR, a microbial adaptive immune system that recognizes and degrades exogenous nucleic acids. In this process, a CRISPR RNA (crRNA) molecule guides the Cas enzyme to a specific target sequence through hybridization. Each crRNA can be custom‐designed to complement a nucleic acid sequence associated with a pathogen or disease marker, ensuring precise targeting. Once guided by the crRNA to a complementary nucleic acid target, the Cas enzyme selectively recognizes and degrades the target DNA or RNA. Some Cas proteins, such as Cas12 and Cas13, exhibit collateral nuclease activity, triggering a cascade of cleavage events that amplify the signal. The amplified signal can then be detected through fluorescent reporters, lateral flow assays, or other biosensors, enabling quantitative detection of the target.
Recent advancements have focused on encapsulating CRISPR/Cas systems within fusogenic liposomes for molecular diagnostics, particularly for analyzing exosomal RNA. Exosomes, a subset of EVs, are 50–200 nm nanoparticles surrounded by a lipid bilayer that transports biomolecules such as RNA, DNA, proteins, and lipids from their parent cells. The encapsulation of CRISPR/Cas components within fusogenic liposomes eliminates the need for RNA extraction, allowing direct analysis of intact exosomal RNA. This innovative approach not only simplifies the workflow but also enhances target specificity and signal amplification, providing a highly sensitive method for the detection of exosome biomarkers.
CRISPR/Cas‐based signal amplification systems integrated with membrane fusion platforms offer remarkable sensitivity and specificity for detecting RNA within exosomes, making them a promising tool for early cancer diagnosis and disease monitoring.[

124

] For instance, Zhou et al. applied CRISPR/Cas13a technology in fusogenic liposomes based on the electrostatic interaction for breast cancer diagnosis. Their system achieved a detection limit of 0.38 fm for exosomal miRNA, distinguishing between early and advanced cancer stages using serum.[

124a

] Similarly, Hong et al. developed an EV miRNA detection platform by encapsulating the CRISPR/Cas13a detection components into a cationic liposome (Figure 5d).[

125

] This fusion system allowed accurate quantification of the number of miRNA‐positive EVs in the tumor‐derived EV subpopulation, which could further increase the difference in plasma EV analysis between cancer and healthy controls. In addition, they showed that a much higher number of EVs are found in the plasma of cancer patients than in healthy controls by capturing epithelial cell adhesion molecule (EpCAM)‐positive EVs through multiplex protein‐miRNA analysis of tumor EVs and quantifying miR‐21‐5p‐positive EVs in the subpopulation.
The CRISPR/Cas‐based signal amplification system is versatile, enabling both nucleic acid and protein analysis. It can also be combined with conventional isothermal nucleic acid amplification methods to establish various signal amplification platforms. Future research should focus on achieving absolute quantification, stabilizing the encapsulation of CRISPR/Cas components (including Cas proteins, crRNA, and probes) within liposomes, improving the stability of liposomes loaded with these reagents, and enabling high‐speed analysis of biomarkers in encapsulated exosomes.

4.2
Controlled Fusion by Specific Triggers
4.2.1
pH‐Sensitive Fusion Systems
pH‐sensitive fusion systems represent a critical approach for controlling membrane fusion events, particularly in diagnostic applications targeting diseased tissues.[

8a

] Biological processes such as endocytosis, viral entry, and intracellular trafficking are inherently pH‐dependent, relying on the acidic environments in organelles such as endosomes or lysosomes (pH 5–6). These physiological variations in pH provide an opportunity to design nanomaterials that selectively undergo fusion in response to specific pH changes. A comparative analysis of viral fusion mechanisms highlights the significance of pH specificity. Sharma et al. revealed that HIV‐1 preferentially fuses with pH‐neutral intracellular vesicles rather than with acidified compartments, contradicting the common assumption that low pH is universally required for viral entry (Figure

6

a).[

126

] This suggests that pH‐neutral targeting strategies may be critical for effectively diagnosing or intercepting HIV‐1 infections. In contrast, Rawle et al. demonstrated that the hemifusion of the Zika virus is initiated by low pH, but the hemifusion rate is relatively independent of pH.[

127

] Their single‐virus kinetic analysis revealed a roughly three‐fold increase in lipid mixing efficiency from pH 6.9 to 4.6 while also showing that fusion rates remain largely pH‐insensitive. This indicates that although pH is sufficient to trigger fusion, it is not the rate‐limiting factor in the process.
These insights suggest a broader design principle, including that pH‐sensitive behavior should be customized to the fusion landscape of each biological context. Yuba et al. operationalized this principle by engineering liposomes modified with protonatable polymers, 3‐methylglutarylated poly(glycidol), which remained stable at neutral pH but destabilized below pH 6 to enable endosomal membrane fusion and cytosolic delivery of antigenic proteins to dendritic cells.[

128

] Their system achieved ≈80% pyranine release at pH 5.0–5.5, indicating robust destabilization promoted by the acidic endosomal condition. Such precision in pH responsiveness directly contributed to the enhancement of cytotoxic T‐cell response in cancer immunotherapy. Building on this, emerging pH‐sensitive fusion designs, such as polymersomes designed to undergo stepwise fusion in response to pH shifts, mimic the staged nature of biological fusion and allow for controlled intracellular delivery and mechanistic studies of endosomal trafficking.[

129

] In parallel, Doyle et al. adopted a different but complementary tactic by demonstrating that transient receptor potential melastatin 7 (TRPM7) ion channels are essential for maintaining the acidic pH of virus‐containing endosomes.[

130

] By sustaining proton pumping and endosomal acidification, TRPM7 indirectly governs the fusion competence of pH‐sensitive systems, highlighting the potential of incorporating channel‐like functionality into synthetic platforms to improve performance and control.
Taken together, these studies converge on a critical insight that robust pH‐sensitive fusion systems require more than just responsiveness to acidity. They demand integrated design approaches that combine structural lipid or polymer elements with biological functionality. Tailoring nanomaterials to accommodate the complexity of pH‐initiated fusion processes can increase the accuracy of biosensing platforms, enable programmable intracellular delivery, and ultimately enhance therapeutic precision across a range of pathological environments.

4.2.2
Ion‐Activated Fusion
Ion‐activated fusion systems exploit the ability of specific ions, such as Ca2⁺, Na⁺, or K⁺, to induce membrane fusion by triggering conformational changes in fusion proteins or lipids.[

131

] In biological systems, ions play a critical role in synaptic vesicle fusion, where the influx of Ca2+ ions prompts vesicles to fuse with neuronal membranes, facilitating neurotransmitter release.[

132

] Inspired by this mechanism, synthetic nanomaterials have been engineered to emulate and manipulate ion‐sensitive fusion processes under physiologically relevant conditions. Tahir et al. designed a gold nanoparticle functionalized with an amphiphilic ligand that mimics natural fusogens by promoting lipid stalk formation.[

133

] Upon Ca2+ addition, these nanoparticles undergo full membrane fusion, reducing the energy barrier for membrane remodeling while offering precise, ion‐tunable control. Similarly, Canepa et al. employed a cholesterol‐containing liposome system decorated with gold nanoparticles to create a minimalistic fusion system responsive to divalent cations such as Ca2⁺ and Mg2⁺.[

134

] Despite differing in architecture, both studies converge on a shared design insight that incorporating ion‐sensing moieties into the structural framework of nanomaterials enables highly selective, controllable fusion behavior that is adaptable to biosensing and delivery applications.
The principle of ion coordination becomes even more nuanced when considering biological fusion proteins. Singh et al. explored the calcium sensitivity of the SARS‐CoV‐2 spike protein, particularly in evolved variants such as D614G and Delta (Figure 6b).[

135

] Their results showed that these spike proteins function as dynamic Ca2⁺ sensors, finely attuned to the calcium‐rich, low‐pH environment of endosomes. In particular, the fusion efficiency, measured by detergent‐normalized, FRET‐based lipid mixing assays, was found to be ≈90% with Delta spike strains at a Ca2+ concentration of 500 µm, reporting the highest value compared with other spike variants of concern. Premature Ca2+ activation led to spike inactivation, demonstrating that effective ion‐activated fusion systems must not only respond to the right ionic cues, but also do so at the right time. This principle is critical for designing diagnostic nanomaterials that should synchronize ion binding with endosomal maturation to avoid off‐pathway or non‐productive fusion.
Extending the concept of ion‐mediated control beyond viral proteins, Kumar et al. developed a chemically programmed exosome fusion platform using Fe3⁺‐mediated catechol coordination to drive controlled membrane fusion between exosomes.[

136

] The system enabled the intracellular assembly of multienzyme nanoreactors, confirmed by orthogonal fusion assays including FRET‐based lipid mixing (≈33% nitrobenzoxadianole fluorescence increase) and cobalt‐calcein and terbium‐dipicoloinic acid signaling (≈36% fluorescence increase). These engineered nanoreactors exhibited sustained catalytic activity, including in situ adenosine triphosphate synthesis in live cells and 3D tissue spheroids, offering a compelling prototype for synthetic organelle design.
Therefore, these studies highlight that ion‐triggered fusion is not merely a biomimetic feature but a design axis that enables modular, condition‐responsive, and temporally coordinated fusion platforms. The ability to integrate ion‐sensitive elements into nanomaterial systems unlocks a versatile toolkit for developing advanced diagnostics. Moving forward, enhancing the temporal and spatial fidelity of these systems will be of importance in achieving high selectivity and functional integration in complex biological matrices.

4.2.3
Enzyme‐Triggered Fusion
Enzyme‐triggered fusion systems offer a strategic advantage in diagnostic design by responding selectively to disease‐associated enzymatic activity rather than relying on passive signals such as pH or ion concentration. These systems enable higher specificity and responsiveness in complex biological environments by linking fusion activation to functional biomarkers. For example, Kim et al. developed a host cell‐mimic polymersome system, termed FluSome, designed to differentiate between highly pathogenic (HPAIV) and low pathogenic avian influenza viruses (LPAIV).[

137

] The system exploits the differential cleavage of HA by trypsin‐ and furin‐like proteases, which are characteristic of LPAIV and HPAIV, respectively. The system demonstrated rapid and accurate discrimination of viruses with 99% accuracy in 30 min, achieving high sensitivity through real‐time FRET‐based detection. Wei et al. also developed enzyme‐responsive liposomes incorporating cleavable ester and peptide bonds for controlled drug release, emphasizing the role of phospholipases and matrix metalloproteinases in mediating liposome destabilization and fusion.[

138

] Compared to passive or constitutively active platforms, these enzyme‐guided designs enable site‐specific release and activation, which is particularly valuable in distinguishing between healthy and pathological tissues. Their findings highlighted the importance of tailoring enzyme‐responsive motifs to the overexpressed enzymes in the disease microenvironment, ensuring targeted release while minimizing off‐target effects.
However, enzymatic control is not limited to fusion initiation. It also shapes the kinetic and mechanistic profile of the entire fusion process. Cervantes et al. examined SARS‐CoV‐2 spike‐mediated fusion and found that while angiotensin‐converting enzyme 2 receptor binding enhances fusion efficiency, it is not strictly necessary under synthetic conditions (Figure 6c).[

139

] This observation challenges assumptions about trigger exclusivity and suggests that enzyme‐ or receptor‐based systems should be supplemented with auxiliary control layers, such as synthetic protease activation or membrane mimicry, to enhance robustness. This layered approach accommodates the redundancy and variability often found in biological systems, enabling diagnostic platforms to remain effective across different physiological and pathological conditions. Building on this view of fusion, Hennig et al. investigated the membrane fusion protein IM30 in cyanobacteria and chloroplasts, revealing a mechanism distinct from classical SNARE‐driven fusion.[

140

] Rather than relying on specific enzymatic triggers, IM30‐mediated fusion was regulated by the presence of Mg2+ ions and local lipid composition, underscoring a vital complement to enzyme‐based systems.
Collectively, these findings indicate that a modular design approach is necessary for next‐generation diagnostic nanomaterials. Instead of relying on a single trigger, future systems should integrate enzyme‐sensitive elements, protease‐cleavable domains, receptor analogs, and membrane‐active features to enhance their functionality. Such multifunctional constructs will not only strengthen selectivity and tunability but also enable adaptation to evolving disease contexts, ultimately improving the versatility, resilience, and precision of membrane fusion‐inspired diagnostic platforms.

4.2.4
Light‐Activated Fusion
Light‐activated fusion provides a non‐invasive, spatiotemporally controlled method for triggering membrane fusion. By incorporating photo‐responsive components, such as photosensitive lipids or peptides, nanomaterials can undergo fusion upon exposure to specific light wavelengths. This approach allows for precise activation in targeted tissues or cells, making it ideal for therapeutic and diagnostic applications. Duan et al. developed a near‐infrared (NIR) light‐activated nucleic acid cascade recycling amplification system for spatiotemporal mRNA imaging in living cells.[

116

] By utilizing upconversion nanoparticles to convert NIR light into visible wavelengths, their system achieved precise nucleic acid activation with high spatial resolution and signal amplification. Similarly, Wang et al. introduced a triplet–triplet annihilation‐based upconversion system for nanoparticle phototargeting, wherein polymeric micelles encapsulating photosensitizer and annihilator molecules facilitated targeted ligand exposure upon light activation.[

141

] This system enabled efficient targeting at low irradiance levels, offering reduced photodamage risks. In another study, Liu et al. designed a photo‐controlled, self‐powered DNA walking machine for intracellular miRNA imaging, utilizing UV light to release DNAzyme‐based walkers along a gold nanoparticle track.[

142

] Key advantages of these studies include precise spatiotemporal control, minimal invasiveness, and enhanced sensitivity; however, limitations such as phototoxicity, stability challenges, and the need for deeper tissue penetration remain critical areas for improvement.
A further refinement of the studies mentioned above, Huang et al. demonstrated the direct application of light‐triggered fusion mechanisms for targeted drug delivery in cancer therapy (Figure 6d).[

116

] They incorporated o‐nitrobenzyl phosphate‐functionalized DNA strands onto liposomal membranes, which underwent light‐induced cleavage to facilitate membrane fusion with target cells via a DNA zipping motion. The use of NIR light enabled deep tissue penetration and minimized collateral damage to healthy cells, offering a controlled, on‐demand release of encapsulated therapeutic agents. This strategy provided precise control over fusion events, reducing systemic toxicity and enhancing therapeutic efficacy. Kong et al. developed a light‐triggered liposomal drug delivery system using photodegradable PEG coatings to control membrane fusion selectively.[

143

] In their approach, light exposure induced the dePEGylation of liposome surfaces, revealing fusogenic peptides that enabled targeted fusion with cancer cells. This system demonstrated enhanced intracellular drug delivery and selective tumor targeting in a zebrafish xenograft model.
Overall, light‐triggered fusion systems present a highly promising platform for biosensing and diagnostics by offering precise, remote‐controlled activation of fusion events. Future research should focus on enhancing biocompatibility, improving light penetration in deep tissues, and developing multi‐wavelength responsive systems to enable broader biomedical applications.

5
Applications
The practical applications of membrane fusion‐inspired nanomaterials are becoming increasingly evident in their potential to revolutionize diagnostic methods for cancer, viral infections, and other diseases. By mimicking the natural fusion process, these nanomaterials facilitate the development of innovative strategies for detecting disease biomarkers with increased sensitivity, specificity, and simplicity. The integration of these nanomaterials with advanced signal amplification techniques, CRISPR/Cas‐based systems, and precise fusion control approaches addresses the key limitations of conventional methods, such as invasiveness, complexity, and insufficient detection sensitivity and selectivity. The subsequent section will provide a detailed examination of the potential of these nanomaterials in two critical applications—virus detection and exosome‐based cancer diagnostics—while highlighting their distinctive advantages and opportunities for further advancement (Table

1
).
Table 1 summarizes five representative types of membrane fusion‐inspired nanomaterials in these contexts: polymersomes, liposomes, lipid‐polymer hybrid nanoparticles, cancer cell‐derived membrane vesicles, and virus‐mimicking fusogenic vesicles. Although these nanomaterials share a common vesicle‐like morphology, comprising a hydrophilic core surrounded by a membrane, they differ in source materials, structural properties, and fusion behaviors. Polymersomes and liposomes are synthetic vesicles composed of amphiphilic block copolymers and phospholipids. Polymersomes possess thicker and more stable membranes with high mechanical strength and tunability, making them well‐suited for diagnostics in prolonged conditions. Liposomes, in contrast, are widely used due to their biocompatibility and ease of functionalization. Their membrane properties can be tailored by introducing cholesterol, charged, or PEGylated lipids to modulate stability and fusogenicity. Lipid‐polymer hybrid nanoparticles combine the unique characteristics of polymeric nanoparticles and PEGylated lipoplexes and feature a core–shell–corona structure, displaying improved structural stability and loading capacity compared to lipoplexes.[

144

] Their lipid surface enhances compatibility with biological membranes and supports fusion‐based interactions.
Cancer cell‐derived or virus‐mimicking fusogenic vesicles represent bio‐derived systems that harness native membrane components. Cancer cell‐derived vesicles preserve the surface proteins and lipids of their parent cancer cells, enabling selective homotypic recognition with cancer‐derived exosomes. This resembles natural intercellular recognition and increases specificity for tumor‐associated targets. Virus‐mimicking vesicles, on the other hand, are engineered from membranes expressing viral fusion proteins, allowing them to emulate a virus‐like entry route and efficiently fuse with target vesicles. Taken together, these nanomaterial systems demonstrate remarkable adaptability across different biological targets, detection conditions, and fusion strategies. By strategically matching their structural and functional attributes to diagnostic objectives, membrane‐inspired nanomaterials continue to advance the performance and clinical applicability of next‐generation biosensing technologies.
5.1
Virus Detection
Membrane fusion‐based biosensors have emerged as a powerful approach for virus detection by mimicking the natural entry pathways of enveloped viruses.[

145

] These biosensors exploit the membrane fusion process to generate quantifiable signals, offering high sensitivity, specificity, and rapid response times compared to conventional diagnostic methods such as quantitative polymerase chain reaction (qPCR) and antigen‐based assays. Recent advancements have focused on leveraging biomimetic and nanostructured materials to enhance detection efficiency and provide functional insights into viral infectivity.
A notable contribution in this field is the membrane rigidity‐tunable fusogenic nanosensors developed by Park et al. for detecting influenza A virus (IAV) (Figure

7a
).[

146

] This system features liposomal membranes with adjustable cholesterol content, allowing fine‐tuning of membrane fusion kinetics to enhance sensitivity and specificity. By optimizing membrane rigidity, the nanosensor achieved efficient detection of IAV in complex biological samples such as avian feces. Unlike conventional detection methods that rely on genetic or protein markers, this system directly assesses viral fusion potential, providing real‐time detection with minimal sample processing. Moon et al. introduced a label‐free and colorimetric detection platform using plasmonic vesicles to identify the IAV.[

147

] This system exploits receptor‐mediated viral fusion with the plasmonic vesicles, leading to a visible color change that enables rapid and straightforward visual detection. The approach offers a simple, equipment‐free alternative to traditional diagnostic methods and is particularly suitable for point‐of‐care applications.
Another notable advancement by Park et al. is the development of a cell‐mimetic biosensor for the detection of avian influenza virus (AIV), which leverages polymeric nanoparticles functionalized with sialic acid, a well‐established viral receptor (Figure 7b).[

148

] This biomimetic strategy facilitates selective fusion between the viral particles and the sensor, leading to the generation of quantifiable fluorescence signals that enable the differentiation between highly pathogenic and low‐pathogenic strains. A key advantage of this system lies in its ability to address limitations associated with conventional diagnostic assays by exploiting variations in furin and trypsin enzyme activity to accurately distinguish between viral strains of differing pathogenicity. The integration of this enzymatic differentiation mechanism enhances the biosensor's specificity and enables rapid, reliable detection, offering a promising approach for effective AIV screening in clinical and field settings.
In SARS‐CoV‐2 detection, Ning et al. developed a liposome‐mediated fusion assay targeting RNA‐positive EVs in plasma (Figure 7c).[

149

] The assay utilizes CD81‐targeting antibodies to capture EVs, which are subsequently fused with reagent‐loaded liposomes containing reverse transcriptase, RPA, and CRISPR‐Cas12a components. This fusion event triggers an ultrasensitive fluorescent signal, enabling highly sensitive detection of SARS‐CoV‐2 RNA. The system successfully detected viral RNA in plasma even when respiratory samples tested negative, providing a valuable tool for identifying systemic viral infections. Chao et al. proposed an innovative cell‐free bioelectronic platform that mimics the biological steps of viral infection by detecting virus binding and membrane fusion through electrical signals (Figure 7d).[

145

] Using SARS‐CoV‐2 as a model, the platform effectively differentiated between Omicron subvariants BA.1 and BA.4 by capturing their distinct fusion signatures. The rapid (≈20 min) and label‐free detection approach of this system offers a significant advantage over conventional methods, providing real‐time, quantitative insights into viral infectivity.
Despite the substantial progress achieved in fusion‐based virus detection technologies, several challenges remain. These include the need for precise environmental conditions to maintain membrane integrity, scalability for high‐throughput diagnostics, and ensuring consistent performance in complex biological matrices. Future research efforts should focus on enhancing the stability of fusogenic materials, integrating multiplexed detection platforms to enable simultaneous identification of multiple viral strains, and developing cost‐effective production methods for commercial viability. Addressing these challenges will be essential to fully leverage the potential of membrane fusion‐based diagnostics for rapid and accurate virus detection across diverse clinical and environmental settings.

5.2
Exosome‐Based Cancer Diagnostics
5.2.1
Exosome Detection
Membrane fusion‐based nanomaterials have emerged as a powerful tool for the detection of exosomes, which are increasingly recognized as critical biomarkers for cancer and other diseases. Exosomes, nanosized EVs secreted by various cell types, carry molecular signatures such as miRNAs and proteins that reflect the physiological and pathological state of their cells of origin. Traditional exosome detection methods often involve multi‐step isolation and analysis techniques such as ultracentrifugation and reverse transcription qPCR (RT‐qPCR), which are time‐consuming and labor‐intensive. In contrast, membrane fusion strategies provide a rapid and efficient alternative, enabling direct and in situ analysis of exosomal cargo with minimal sample processing.
To address these limitations, membrane fusion‐inspired fusogenic nanoparticles have emerged as a promising tool for cancer biomarker detection. These systems, particularly when integrated with liquid biopsy approaches, offer significant advantages, including non‐invasiveness, minimal sample volume requirements, cost efficiency, reduced analysis time, and improved specificity and sensitivity. Liquid biopsies, which analyze bodily fluids such as blood or urine, enable cancer detection by isolating and studying EVs, including exosomes, which carry cancer‐associated biomarkers like nucleic acids and proteins. Researchers have developed innovative strategies using fusogenic nanoparticles to encapsulate signal amplification probes and trigger fusion between exosomes and lipid nanoparticles.
Park et al. developed an all‐in‐one fusogenic nanoreactor (FNR) encapsulating DNA‐fueled molecular machines for the rapid and direct detection of exosomal miRNAs associated with breast cancer (Figure

8

a).[

112a

] This system leverages fusogenic liposomes that selectively interact with cancer‐derived exosomes and induce membrane fusion under specific conditions, such as low pH and protease activation. The FNRs efficiently detected target miRNAs within 30 min, achieving an accuracy of 86.4% in distinguishing breast cancer patients from healthy donors and an improved diagnostic accuracy of 95.4% when analyzing multiple miRNAs simultaneously. The FNR platform offers a simplified workflow with high throughput and minimal hands‐on time compared to conventional RT‐qPCR‐based methods.
Unlike general approaches that use cationic liposomes, which increase the likelihood of non‐specific fusion with untargeted cells or EVs, targeted diagnostics rely on surface functionalization strategies to ensure selective fusion. These strategies, including zipper DNA‐mediated fusion, homotypic recognition, and dual‐surface‐protein‐guided orthogonal labeling, simplify the detection process while improving specificity and sensitivity. Xie et al. introduced a DNA zipper‐mediated membrane fusion approach for the rapid detection of exosomal miRNAs, particularly miR‐21, a well‐established cancer biomarker (Figure 8b).[

113g

] Their system utilized lipid vesicle probes loaded with molecular beacons and functionalized with zipper DNA constructs (ZDCs) on the surface. Upon interaction with complementary ZDCs on exosomes, a zipper‐like DNA hybridization process facilitated membrane fusion, triggering fluorescence‐based miRNA detection within 30 min. This approach demonstrated high specificity and sensitivity in distinguishing cancer‐derived exosomes from normal counterparts, with successful application to both cell culture and patient serum samples. Compared to traditional RNA extraction‐based methods, this system offers a non‐destructive and rapid detection alternative.
Furthermore, Lei et al. developed the dual‐surface‐protein‐guided orthogonal recognition (SORTER) system for simultaneous subset tracing and miRNA profiling of tumor‐derived exosomes (Figure 8c).[

150

] This one‐pot assay employs aptamer‐based labeling of tumor‐specific markers (CD63 and EpCAM) to selectively identify cancer‐derived exosomes, followed by targeted membrane fusion with liposome probes for in situ miRNA detection. The system demonstrated an exceptional diagnostic accuracy of 100% for prostate cancer and 90.6% for metastatic disease classification. The SORTER platform outperforms conventional exosome analysis methods by enabling multiplexed miRNA detection without extensive sample processing.
Zhuo et al. further enhanced cancer diagnostics by developing a system that uses three quantum dots with distinct emissions, labeled with antibodies and encapsulated within fusogenic vesicles.[

151

] This system enables multiplexed detection of exosomal miRNAs with ultrahigh sensitivity and provides insights into cancer stage monitoring based on biomarker expression levels. Similarly, Cao et al. utilized camouflaged catalytic DNA machinery and homotypic recognition to trigger fusion between cancer exosomes and cancer cell membrane vesicles.[

124b

] Their electrochemical signal‐based approach demonstrated a positive correlation between signal intensity and disease progression, providing a valuable tool for breast cancer stage monitoring.
Yang et al. proposed a silica nanoquencher‐based nanoplatform with liposome encapsulation for the rapid detection of exosomal miRNAs (Figure 8d).[

152

] This system relies on the fusion of liposome‐encapsulated silica nanoparticles with exosomes, delivering fluorescence‐labeled antisense oligonucleotides for a switchable “off‐on” detection mechanism. The assay successfully differentiated tumor‐related exosomes from normal ones without the need for RNA extraction or amplification, demonstrating its potential as a simple and rapid detection platform applicable to clinical liquid biopsy.
Collectively, these membrane fusion‐based strategies represent significant progress in exosome detection for disease diagnosis. They provide rapid, label‐free, and amplification‐free approaches that streamline traditional workflows and enhance diagnostic accuracy. However, challenges such as achieving consistent fusion efficiency across different exosome populations, enhancing specificity, and ensuring scalability for clinical implementation must be addressed. Future research should focus on refining targeting mechanisms, improving biosensor stability in biological fluids, and integrating these systems into portable diagnostic platforms to facilitate their widespread adoption in clinical practice.

5.2.2
Exosome Detection at the Single‐Particle Level
Recent advancements in cancer diagnostics have focused on integrating cutting‐edge technologies into exosome detection platforms, leveraging the biological significance of exosomes as carriers of disease‐specific biomarkers. Among these developments, membrane‐fusion‐inspired nanomaterials have emerged as transformative tools by mimicking natural biological processes to enable targeted and efficient detection of exosome‐associated biomarkers at the single‐particle level. These nanomaterials provide enhanced sensitivity, specificity, and scalability for high‐throughput cancer screening, offering significant potential to overcome traditional diagnostic limitations such as low sensitivity, time‐consuming workflows, and the need for invasive sampling methods. By integrating these systems with advanced technologies like total internal reflection fluorescence (TIRF) microscopy,[

153

] flow cytometry,[

113
,
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] and droplet digital PCR,[

154

] researchers have unlocked the ability to simultaneously detect multiple biomolecules, such as proteins, miRNAs, and mRNAs, within individual exosomes or EVs. Such advances provide comprehensive insights into exosome or EV phenotypes, improving the precision of disease diagnosis, prognosis, and treatment monitoring.
In this regard, Zhou et al. developed a high‐throughput nano‐bio chip integrated system for liquid biopsy (HNCIB) capable of analyzing single EVs to simultaneously detect proteins and nucleic acids such as mRNA and miRNA (Figure

9

a).[

153

] This HNCIB platform combined a high‐throughput nano‐biochip for efficient EV capture with TIRF microscopy for rapid, high‐resolution detection. The integration of a convolutional neural network deep learning algorithm enabled automated analysis of complex image data, quantifying the distribution of mRNA, miRNA, and membrane proteins with high accuracy. Using this system, Zhou et al. demonstrated the ability to detect programmed death ligand 1 (PD‐L1) and miR‐21 expression in EVs derived from lung adenocarcinoma patients, allowing the quantification of CD63+ and PD‐L1+ EV subpopulations. This capability facilitates not only monitoring PD‐L1 levels during immunotherapy but also identifying patient populations likely to benefit from such treatments based on the proportions of PD‐L1+ EVs and other associated biomarkers.
Similarly, Xu et al. developed a dual CRISPR‐Cas‐based single‐EV evaluation system capable of detecting both EV surface proteins and internal miRNAs at the single‐molecule level (Figure 9b).[

155

] The system employs Cas12a to detect surface proteins through antibody‐DNA conjugates, translating protein signals into DNA signals, while Cas13a analyzes internal miRNAs via membrane fusion between EVs and liposomes. Integrated into a microfluidic chip for digital analysis, this platform enabled fluorescence‐based quantification of miRNA/protein‐positive EVs with exceptional precision, achieving an absolute detection capability of 214 EVs µL−1. Xu et al. successfully used the system to distinguish breast cancer patients from healthy donors with an impressive accuracy of 92%, highlighting its potential for precise cancer diagnostics.
Moreover, Feng et al. introduced an encoded fusion‐mediated strategy for miRNA signature profiling of tumor‐derived EVs, specifically targeting pancreatic cancer diagnosis (Figure 9c).[

156

] This system utilizes encoded‐targeted‐fusion beads (ETFBs) that selectively recognize and fuse with EVs. By employing molecular beacons for miRNA quantification and barcoded signals for flow cytometry‐based identification, the ETFB system enables multiplex detection of six pancreatic cancer‐associated miRNAs from minimal plasma samples (2 µL) without requiring EV isolation or lysis. The approach achieved a remarkable diagnostic accuracy of 98%, differentiating pancreatic cancer patients from those with pancreatitis and healthy donors within a short processing time of 2 h. This rapid, multiplexed analysis offers significant advantages for early cancer detection and monitoring.
In another development, Clarissa et al. reported EV‐CLIP, a droplet‐based digital platform designed for profiling EV‐associated RNAs directly from unprocessed blood plasma (Figure 9d).[

154

] EV‐CLIP relies on charged‐liposome‐induced fusion (CLIP) within a microfluidic chip, enabling the specific and efficient detection of exosomal mRNAs and miRNAs without extensive sample preprocessing. The optimized surface charge of CLIPs ensures targeted fusion with EVs, eliminating the need for isolation or RNA extraction. The platform demonstrated exceptional diagnostic accuracy in detecting EGFR L858R and T790M mutations in lung cancer patients, achieving area under the curve values of 1.0000 and 0.9784, respectively. Furthermore, EV‐CLIP enabled real‐time monitoring of these mutations during chemotherapy, showcasing its potential for precise and dynamic disease tracking. However, further optimization of lipid composition and stability is needed to broaden its application across various EV subtypes and clinical environments.
In conclusion, membrane‐fusion‐inspired nanomaterials have paved the way for transformative advancements in single‐EV analysis for cancer diagnostics. These platforms combine non‐invasive, highly sensitive, and multiplexed capabilities to detect tumor‐derived biomarkers, facilitating early diagnosis, treatment monitoring, and improved patient management. Future research should aim to enhance the robustness, scalability, and clinical compatibility of these systems while addressing challenges such as long‐term stability, cost‐efficiency, and broader applicability to diverse cancer types and clinical settings.

6
Conclusion and Outlook
Membrane fusion‐inspired nanomaterials have emerged as a promising advancement in biomedical applications, particularly in diagnostics, by mimicking natural membrane fusion processes to facilitate precise and controlled interactions with target biomaterials. Key insights from this review highlight the pivotal role of biomimetic strategies in enhancing the specificity and sensitivity of diagnostic platforms. The incorporation of fusogenic lipids, fusion peptides, and DNA‐based triggers has enabled the development of nanoscale systems with remarkable control over membrane interactions. Moreover, the integration of advanced signal amplification techniques, such as isothermal nucleic acid amplification and CRISPR/Cas systems, has significantly improved the detection capabilities for infectious diseases and cancer biomarkers. These innovations underscore the potential of membrane fusion‐inspired nanomaterials to revolutionize biosensing, diagnostic precision, and personalized medicine.
Despite the remarkable progress achieved, several challenges remain in the widespread adoption of membrane fusion‐inspired nanomaterials. One major challenge lies in the reproducibility and scalability of these systems, as the complexity of lipid compositions and functionalization processes often results in variability in performance. Additionally, the stability of fusogenic nanomaterials in physiological conditions presents another significant hurdle, as interactions with serum proteins and other biological components can impact their efficacy. The specificity of membrane fusion events also requires further optimization to minimize off‐target effects and unintended interactions. Furthermore, regulatory and translational hurdles pose additional barriers to clinical implementation, requiring extensive validation and standardization to ensure safety and efficacy in real‐world applications. Addressing these challenges is crucial for advancing the clinical utility of membrane fusion‐inspired nanomaterials.
In addition to these challenges, ensuring the biocompatibility of membrane‐inspired vesicles and fusion motifs is essential, especially for in vivo applications. While most diagnostic strategies focus on biomarker detection in excreted biofluids, where immunogenicity risks are minimal, the use of these systems in animal or human models for targeted imaging or biosensing requires detailed assessments of cytotoxicity and immune responses. These concerns become more pronounced for therapeutic applications, where repeated dosing or systemic exposure could lead to adverse effects.[

157

] Future studies should include early‐stage assessments of immune responses and biological safety to ensure the responsible development of fusion‐based nanomaterials.
Although numerous membrane fusion‐inspired diagnostic systems have demonstrated strong analytical performance using clinical samples, none have yet advanced to regulated clinical trials. This translational gap reflects several unmet needs: the absence of scalable, standardized production methods, a lack of consensus around performance metrics for fusion efficiency and membrane stability, and underdeveloped regulatory frameworks for diagnostic approval. Furthermore, most existing systems do not yet satisfy the World Health Organization's REASSURED criteria, which emphasize real‐time connectivity, ease‐of‐use, affordability, and end‐user deliverability,[

158

] further limiting their real‐world deployment. Bridging these gaps will require integrating membrane fusion principles with simplified, robust, and portable diagnostic platforms. Additionally, cross‐sector collaborations will be vital for establishing material standards, validating performance in multi‐center studies, and defining regulatory pathways to enable clinical translation.
Furthermore, there is a growing need for tools that can support the visualization of intracellular trafficking and intercellular exchange of membrane‐inspired vesicles. Choi et al. reported a pH‐responsive boron dipyrromethene‐based probe capable of tracking endogenously secreted EVs from living cells by exploiting the acidic lumen of intracellular organelles.[

159

] While this probe showed promise for imaging natural EV dynamics, its applicability to vesicles with synthetic membranes remains limited due to differences in vesicle origin and pH responsiveness. Nonetheless, this study illustrates the broader value of developing vesicle‐specific imaging probes. Further efforts in molecular probe development will be critical for elucidating the dynamic behavior of fusion‐based vesicles in complex biological environments.
The future of membrane fusion‐inspired nanomaterials offers exciting opportunities for innovation and expansion across various biomedical fields. Advances in lipid engineering, surface functionalization, and modular system design will enable the development of next‐generation nanomaterials with improved stability, selectivity, and tunability. The convergence of artificial intelligence and machine learning with nanotechnology could provide powerful tools for optimizing nanomaterial design and predicting their interactions with biological systems. Additionally, the exploration of multi‐functional and stimuli‐responsive nanomaterials will unlock new possibilities for theranostics, combining diagnostic and therapeutic functionalities in a single platform. Collaboration between interdisciplinary fields, including materials science, bioengineering, and clinical medicine, will be instrumental in overcoming current limitations and accelerating the translation of membrane fusion‐inspired nanomaterials into clinical practice. Ultimately, these efforts will shape the emergence of diagnostic tools that are more personalized, effective, and accessible for diverse biomedical challenges.

Conflict of Interest
The authors declare no conflict of interest.