Dual-responsive coloration of Janus droplets via total internal reflection and interference applied as single-use freezing indicators

Domain: chemistry
Published: 2026-02-27
Editorial outcome: minor_revision

Abstract
Responsive optical materials offer real-time visual feedback to external stimuli by converting them into visible light signals, holding great promise in display, sensing, and information encryption. Typically, heterogeneous Janus droplets can exhibit vivid coloration induced by total internal reflection and interference (TIRI) at concave liquid–liquid interfaces. Yet, achieving stimuli-responsive TIRI-based structural colors remains a challenge due to the difficulty in controlling interface curvature and reflective behavior under external stimuli. Here, we report the fabrication of Janus droplets with tunable TIRI structural colors that respond to both surfactant composition and temperature. By tailoring surfactant formulation, the droplet morphology and interface curvature can be adjusted, enabling reversible modulation of structural color appearance, including both iridescent and non-iridescent modes depending on incident light conditions. Furthermore, the structural color can be reversibly or irreversibly switched on/off in response to temperature changes, due to phase transitions that alter light reflection at the concave interface. Owing to their temperature-dependent optical response and real-time visual signaling, these droplets can serve as single-use security labels for monitoring freeze history of freeze-sensitive pharmaceuticals, providing reliable indication of product integrity.

Introduction
Structural coloration arises from the interactions of light with nano/micro structures through mechanisms such as diffraction, reflection, and interference1, 2, 3, 4, 5, 6. In nature, after millions of years of evolution, structural colors are widespread in plants and animals, playing an important role in their predation, mating, and camouflage, due to their vividness and durability7, 8, 9, 10, 11, 12, 13. Unlike chemical pigments, which rely on selective absorption or emission of light, structural colors are physically generated and are thus known for their brightness, environmental friendliness, high chromatic purity, and excellent resistance to fading14, 15, 16, 17, 18, 19.

With advances in the design of artificial micro- and nanostructures, the fabrication and tuning of structural color materials have progressed rapidly. In particular, stimuli-responsive structural color materials that can change their colors in response to external stimuli have been prepared using smart materials20, 21, 22, 23, 24, providing real-time visible feedback to external stimuli such as temperature, magnetic field, pressure, solvent, light, electric field, pH, and others25, 26, 27, 28, 29, 30. In the field of thermal-responsive color change, the integration of thermally sensitive structural-color materials with flexible heaters enables full-spectrum reversible color modulation in soft robots, offering a strategy for biomimetic camouflage and human–machine interaction31, 32. Regarding mechanical response, interference lithography based on photopolymerizable elastomers exploits strain-induced deformation of periodic photonic nanostructures to achieve precise optical tuning33, 34. This approach facilitates high-sensitivity mechanical sensing and real-time visual monitoring of deformation, meeting the demands of wearable devices and intelligent structural health monitoring. Owing to their immediate and perceptible optical response, such materials show great potential in advanced displays, anti-counterfeiting, chemical and physical sensing, and environmental monitoring35, 36, 37, 38.

Conventionally, the generation of structural color requires highly ordered nano/micro structures. Top-down approaches such as photolithography allow precise fabrication while demand costly instrumentation and complex procedures. Bottom-up self-assembly approach, while more scalable, remains challenging in achieving high uniformity of building blocks and the efficient fabrication of large-area, long-range ordered structures. Notably, in recent years, several strategies have been developed to address these challenges, including roll-to-roll deposition technology39, shear-induced self-assembly40, 41, and template-directed self-assembly42. Several of these technologies have progressed toward commercialization, markedly advancing the practical deployment of structural color materials.

Unlike traditional approaches that rely on long-range ordered structures, an alternative strategy has recently emerged, employing total internal reflection and interference (TIRI) at concave liquid–liquid interfaces within biphasic emulsion droplets to generate structural color43. By carefully tuning the interface curvature inside the biphasic droplet, the incident light undergoes complete internal reflection at the interface and subsequently interferes near the three-phase contact line. As a result, the structural colors are generated and can be modulated by tailoring the droplet size, eliminating the dependency on periodic array structures. Importantly, this method circumvents the need for nanofabrication tools and provides a lithography-free route to producing vivid optical effects. Yet, since TIRI structural colors rely primarily on precise control of interfacial curvature, achieving multiple colors necessitates droplets of different sizes. This limitation is particularly restrictive in anti-counterfeiting and detection applications, where conventional TIRI systems exhibit only monochromatic, static, and unimodal color. Such constrained optical behavior hinders their utility in stimuli-responsive color-changing applications, including smart displays and high-security anti-counterfeiting. Thus, it is highly desirable to develop TIRI-based systems with dynamic multicolor response under simple external-field modulation for advanced optical detection and secure labeling.

Here, we report a dual-stimuli-responsive TIRI structural color system based on monodisperse Janus droplets with tunable concave liquid-liquid interfaces. The structural color can be dynamically modulated by either surfactant composition or temperature, enabling reversible color switching via surfactant-induced morphological reconfiguration of droplets and temperature-triggered phase transitions of droplets. By integrating these dual-responsiveness properties, we demonstrate a real-time cold-chain temperature monitoring label tailored for freeze-sensitive pharmaceuticals. This monitoring system provides dual-range temperature detection, exhibiting reversible color changes near 4 °C and irreversible color loss upon exposure to sub-zero temperatures (< 0 °C), attributed to phase-transition-induced disruption of internal reflection pathways. This system demonstrates good stability and reusability after 20 surfactant/temperature responsive cycling tests, while addresses the limitations of conventional monitoring labels, such as poor sensitivity, narrow detection range, and limited reliability44. Moreover, this platform enables clear visual and irreversible indication of freezing events, offering a practical solution for robust cold-chain integrity tracking.

Results
Fabrication and optical behavior of Janus droplets
Biphasic Janus droplets were fabricated using an emulsion–solvent evaporation approach (Fig. 1a). Typically, a 1:1 mixture of hexadecane (density ρ = 0.77 g mL−1, refractive index n = 1.43) and methoxyperfluorobutane (HFE-7100, ρ = 1.53 g mL−1, n = 1.30) were dissolved in chloroform (50 vol.%). Then, the solution was used as oil phase to prepare monodisperse emulsion droplets by a capillary microfluidic apparatus, with an aqueous solution of Capstone FS-30 and sodium dodecyl sulfate (SDS) serving as the continuous phase (total surfactant concentration: 5 mg mL⁻¹, SDS weight fraction, f = 0.30). Upon chloroform evaporation, the droplets spontaneously underwent liquid-liquid phase separation, forming biphasic Janus droplets with an upward concave hexadecane/HFE-7100 interface (Fig. 1b-f).

When being illuminated, incident light entering the droplet can undergo total internal reflection at the curved hexadecane/HFE-7100 interface if the angle of incidence exceeds the critical angle of θ = 65.4° from arcsin(n /n ) (Fig. 1g). Due to the curved geometry, light rays reflect multiple times at the interface, and their path-length differences generate interference patterns localized near the three-phase contact line (hexadecane/HFE-7100/water), resulting in vivid TIRI structural colors. As shown in Fig. 1e, the Janus droplets exhibit a bright colored ring under directional white light. The droplets with an average diameter of 60.5, 48.2, and 41.6 μm display pink, green, and blue colors, respectively (Fig. 1h-j and Supplementary Fig. 1), confirming that the color depends on droplet size and thus optical path difference.

Interfacial tension and morphological control
The morphology of the droplets and consequently the TIRI structural color are determined by the balance of interfacial tensions among hexadecane/HFE-7100 (γ ), HFE-7100/water (γ ), and hexadecane/water (γ ). As shown in Fig. 2a, a core–shell morphology is formed when γ - γ ≥ γ (HFE-7100-in-hexadecane), or γ - γ ≥ γ (hexadecane-in-HFE-7100). Only when |γ - γ | < γ is the Janus configuration energetically favored. Notably, since γ is extremely low (0.93 mN m-1), the droplet morphology is highly sensitive to small changes in γ and γ 46, 47. This sensitivity provides a means to dynamically regulate the structural color via interfacial tension modulation.

The interfacial tensions, γ , γ and γ , were measured as a function of the weight fraction of SDS (f ) in the FS-30/SDS surfactant mixture (Supplementary Fig. 2 and Supplementary Table 1). The corresponding droplet morphologies are shown in Fig. 2b c. When f < 0.2, the |γ - γ | exceeds γ , leading to core–shell droplets with no visible structural color. Otherwise, Janus droplets are formed. Generally, Janus droplets formed with 0.2 ≤ f ≤ 0.35 exhibit bright TIRI color (Fig. 2c). When f > 0.35, the curvature is insufficient to achieve the critical angle for TIR, and structural color is no longer observed. It is noteworthy that the curvature is not sufficient even for f = 0.30 and 0.35 because the maximum geometric angle near the triple line measured from the vertical is smaller (52.5° and 60.7°) than the critical angle (65.4°) for TIR of hexadecane/HFE-7100 interface. Nevertheless, the refraction of light at water/hexadecane increases the incident angle to the hexadecane/HFE-7100 interfaces, providing TIR.

Effect of droplet size, curvature and illumination
The TIRI structural color of Janus droplets exhibits a strong dependence on droplet size. As shown in Fig. 3a, increase of droplet diameter from 40.3 to 62.7 μm results in a progressive shift in the interference color, spanning a full color cycle from violet to red. This shift originates from changes in the optical path length differences within the droplets: larger diameters have longer propagation lengths between internal reflections, leading to larger path differences and thus red-shifted interference peaks. When concentrated, the emulsions form ink-like dispersions with vivid colors under directional lighting (Fig. 3b). The reflectance spectra reveal narrowband peaks (Supplementary Fig. 3), indicative of the monodispersity and high color purity of the droplets. The CIE chromaticity coordinates form a closed loop, as the emulsion droplets span the full range of rainbow colors (Fig. 3c).

The structural color can also be finely tuned by altering the curvature of the internal interface. This is achieved by adjusting the volume ratio of hexadecane to HFE-7100. As the volume ratio increases from 1:9 to 6:4, the upward concave interface gradually flattens, leading to a reduced curvature radius (Supplementary Fig. 4). This geometric change alters the effective incidence angle and optical path difference, resulting in a continuous color shift.

In addition to geometrical parameters, the observed TIRI color is strongly dependent on illumination conditions. Under ambient light, the droplets appear nearly colorless due to insufficient light intensity for generating noticeable interference (Supplementary Fig. 5a). In contrast, under directional illumination such as a cellphone flashlight, the droplets exhibit brilliant structural colors (Supplementary Fig. 5b). Moreover, both the angle of incident light (α) and the angle of observation (β) influence the observed color (Fig. 4). As shown in Fig. 4a and e, under non-coaxial illumination (α ≠ β), a penguin pattern with droplet diameter of 44.2 μm exhibits a gradual color change from cyan to violet as α increases from 0° to 30° while β is kept at 0°. In contrast, under coaxial illumination (α = β), the structural color remains unchanged. As shown in Fig. 4b and f, the “HUST” pattern remains unchanged when α and β synchronously increase from 0° to 30°. This can be attributed to the spherical symmetry of the Janus droplets that leads to identical interferometric conditions when the illumination and viewing directions are coaxial. In addition, under fixed incident angle (α = 0°), rotating the azimuthal viewing angle (φ) from 0° to 360° does not alter the perceived color (Fig. 4c and g), again due to axial symmetry. However, when the incident light is applied obliquely (α = 30°), the structural color varies with φ (Fig. 4d and h), exhibiting change of hues from pink to orange, green, and violet. Therefore, the Janus droplets exhibit both iridescent and non-iridescent properties depending on the conditions of illumination light and observation, making them a promising candidate for various applications, including display and anti-counterfeiting.

Stimuli-responsive switching of TIRI structural color
Beyond static color modulation, the Janus droplets exhibit dynamic and reversible switching of their TIRI structural color in response to external stimuli, including surfactant composition and temperature. These properties make them promising candidates for intelligent display and anti-counterfeiting applications.

Surfactant-Responsive Switching: The structural color can be reversibly turned on and off by altering the surfactant composition (the total surfactant concentration was maintained at 5 mg mL-1), which modulates the interfacial tensions and thereby the droplet morphology (Fig. 5a-e). For example, the blue-colored Janus droplets lose their structural color upon the addition of Capstone FS-30 to decrease f to 0.10 (Fig. 5b, c). In this case, the γ is preferentially reduced45, resulting in the transition of droplets from a Janus structure to a core-shell structure (hexadecane-in-HFE-7100). Upon reintroduction of SDS to recover f = 0.30, γ is reduced, restoring the Janus morphology and thereby reactivating the TIRI structural color (Fig. 5d). Reflectance spectra confirm that the peak wavelength ( 485 nm, Fig. 5f) remains stable over a single switching cycle (Fig. 5g). To further validate the stability of the Janus droplets under surfactant switching cycling, we performed 20 switching cycles (Supplementary Fig. 6, 7). The results show that the reflectance intensity remained virtually unchanged even after 20 cycles, demonstrating repeatability and structural integrity.

Temperature-Responsive Switching: The droplets also display rapid and reversible thermal responsiveness (Fig. 5h-n). Droplets with diameters of 44.1 μm show vivid blue structural color at 25 °C (Fig. 5i, j), which disappears upon cooling to 4 °C (Fig. 5k, l). This change arises from the phase transition of liquid hexadecane into a polycrystalline solid below its freezing point (18 °C), which decreases droplet transparency and volume. As a result, incident light randomly scatters, preventing the light propagation in the solidified hexadecane droplet, losing colors. Upon warming, the hexadecane reverts to the transparent liquid state, restoring the structural color. The corresponding reflectance spectra show disappearance and reappearance of the 487 nm peak (Fig. 5m), and the system maintains responsiveness and stability over 20 thermal cycles (Fig. 5n). Although a small number of Janus droplets underwent aggregation with increasing cycle numbers, leading to a gradual attenuation in reflectance spectrum intensity (Supplementary Figs. 8, 9), the reflectance spectrum intensity had slightly decreased by less than 20% after 20 cycles.

These dual-responsive and reversible switching mechanisms, based on morphological reconfiguration and phase transition, offer a platform for designing optical tags with real-time, stimuli-triggered visual feedback.

Application in security labels for freeze-sensitive drugs
Despite extensive development, existing temperature indicators for pharmaceuticals (e.g., vaccines and medicines) often suffer from poor sensitivity, narrow operating temperature windows, and limited reliability, significantly restricting their use in cold-chain logistics26, 44. To address these limitations, we leverage the dual-responsive behavior of Janus droplets to construct a visual, single-use security label capable of recording temperature excursions. As shown in Fig. 6, two temperature-monitoring labels were designed by embedding Janus droplets into microgrooves on the surface of pharmaceutical vial caps. These were tailored to respond at two key storage temperatures: 4 °C and −2 °C, covering conditions relevant to human papillomavirus (HPV) vaccines, insulin, and other temperature-sensitive pharmaceuticals.

For the reversible monitoring system (Fig. 6b), the labels exhibit bright orange structural color at room temperature (25 °C) under directional illumination, with a reflectance peak at 605 nm. Upon storage at 4 °C, hexadecane (freeze point: 18 °C) in the droplets solidifies, causing the TIRI color to vanish due to loss of transparency of droplets and thus their internal reflection (Supplementary Movie 1). Upon reheating to 25 °C, the structural color of the label is fully restored (Supplementary Movie 2). Notably, this reversible process is independent of the heating and cooling rates (Supplementary Movies 3, 4), confirming the phase transition mechanism.

In contrast, the irreversible monitoring system is realized by exposing the security labels to sub-zero temperatures (Fig. 6c). When stored at −2 °C, both the hexadecane drops and continuous water phase transform to solid state, while the HFE-7100 phase (freeze point: -137 °C) remains as a liquid state. In this case, the formation of ice crystals destabilizes the HFE-7100/water interface. During thawing, the HFE-7100 phase coalesces (Supplementary Movie 5), causing irreversible structural disruption of the Janus droplets and permanent loss of TIRI color48, 49, 50. This irreversible behavior provides a visual and reliable record of freezing events, ensuring integrity tracking for cold-chain pharmaceuticals.

To better simulate real-world application scenarios, we evaluated mechanical stability and long-term stability of the labels. The results demonstrated that the structural color remained largely unchanged after gentle shaking (Supplementary Movie 6 and Supplementary Fig. 10) and being dropped from a height of 30 cm (Supplementary Movie 7 and Supplementary Fig. 11). Additionally, long-term stability tests showed that the label retained its vivid structural color after 2.5 years of storage at room temperature (Supplementary Fig. 12), and its color could still be fully restored after 15 days of storage at 4 °C (Supplementary Fig. 13). These findings collectively confirm the label's overall stability. This integrated platform overcomes the key limitations of conventional indicators by offering multistage, visually interpretable, and irreversible optical signals, providing a robust solution for real-time quality assurance in freezing-sensitive pharmaceuticals transportation and storage.

Methods
Materials
Hexadecane (purity 99%) and methoxyperfluorobutane (HFE-7100, purity 98%) were purchased from Aladdin. Sodium dodecyl sulfate (SDS, purity > 99%) was obtained from Sigma-Aldrich, and Capstone FS-30 (purity 25%) was purchased from Shanghai Kingpont Industrial Co., Ltd. All chemicals were used as received without further purification.

Fabrication of the Janus droplets
Typically, 1 mL of hexadecane and 1 mL of HFE-7100 were dissolved in 2 mL of chloroform to prepare the oil phase. Monodisperse emulsion droplets were then generated using a capillary microfluidic device, with the oil solution as the dispersed phase and an aqueous solution of Capstone FS-30 and SDS as the continuous phase (SDS weight fraction in mixed surfactants: f = 0.30, total concentration: 5 mg mL-1).

The microfluidic device was consisted of two tapered cylindrical glass capillaries (inner diameter: 0.70–0.75 mm; outer diameter: 0.90–0.95 mm) coaxially aligned within a square capillary (inner diameter: 1.05 mm; outer diameter: 1.50 mm). One cylindrical capillary was sanded to form a 50 μm orifice, referred to as the injection capillary; the other was sanded to 120 μm. The two cylindrical capillaries were aligned tip-to-tip with a spacing of 80 μm inside the square capillary.

The chloroform-based oil solution was injected through the injection capillary as the inner phase, while the mixed aqueous surfactant solution was simultaneously injected from the same direction through the annular space between the injection and square capillaries, serving as the continuous phase. The flow rate of the inner phase was set to 5 μL min-1, while the outer flow rate ranged from 300 to 700 μL min-1. The resulting droplets were collected from the outlet and incubated in the same aqueous surfactant solution at room temperature for 4 days to allow complete evaporation of chloroform.

Preparation of surfactant solutions
Preparation of 0.50 wt.% SDS solution
Weigh 0.50 g of SDS powder and transfer it into a conical flask. Add 100 mL of deionized water, and perform ultrasonication until the SDS is completely dissolved. Then shake the mixture thoroughly to ensure homogeneity.

Preparation of 0.50 wt.% Capstone FS-30 solution
Pipette 2 g of 25 wt.% Capstone FS-30 stock solution into a conical flask. Add 100 mL of deionized water and shake the mixture thoroughly to achieve homogeneity.

Preparation of mixed surfactant solution
The mixed surfactant solution was prepared by combining the aforementioned 5 mg mL-1 SDS solution and 5 mg mL-1 Capstone FS-30 solution in varying ratios. The composition of the mixture is characterized by the mass fraction of SDS, denoted as f , which represents the proportion of SDS in the total surfactant content.

Surfactant-responsive behavior of Janus microparticles
The as-prepared Janus microparticles were dispersed in 1 mL of mixed surfactant solution with f = 0.3, under which condition they exhibited blue structural color resulting from total internal reflection. Subsequently, 2 mL of 5 mg mL-1 Capstone FS-30 solution was added to the system, leading to a structural transition of the microparticles from Janus to core–shell type, accompanied by the disappearance of the structural color. Further addition of 0.86 mL of 5 mg mL-1 SDS solution restored the Janus structure and the structural color. Upon introducing another 7.74 mL of 5 mg mL-1 Capstone FS-30 solution, the microparticles reverted to the core–shell structure, and the structural color vanished again. This process is reversible and can be repeated cyclically, while the total surfactant concentration is maintained at 5 mg mL-1 throughout the experiment.

Characterization
Optical reflection micrographs were acquired using an optical microscope (H600L, Nikon) equipped with a CCD camera (SD1600AC, IMG). Digital photographs were taken with a Canon 200D camera. Interfacial tension was measured using an optical contact-angle measuring instrument (JC2000C1, Dataphysics Instruments, Shanghai Zhongchen Digital Technic Apparatus Co., Ltd.). Reflectance spectra were recorded with a fiber-coupled spectrometer (USB4000, Ocean Optics).

Data availability
The data that support the findings of this study are available from the corresponding authors upon request. Unprocessed raw data are provided as Supplementary Data 1. Source data are provided with this paper.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript presents a Janus biphasic droplet platform that generates structural color via TIRI at concave liquid–liquid interfaces. High-purity structural colors are achieved without solid patterning, and the system exhibits dual-mode responsiveness to surfactant composition and temperature. Overall, the concept is novel and broadly applicable. Thus I recommend its publication as a full paper after addressing the following issues:\n1.Compared with traditional cold-chain indicators, what advantages does a structural-color readout offer, and what are the key translation challenges? In addition, relative to other structural-color-based cold-chain indicators, what specific advantages does the present work provide?\n2.The authors state, “Upon reheating to 25 °C, the structural color of the label is fully restored.” Will this performance degrade with an increasing number of cooling–heating cycles under practical conditions?\n3.For the reported “irreversible color loss upon exposure to sub-zero temperatures (< 0 °C),” does the irreversibility also depend on storage duration at sub-zero temperatures? Please provide supporting data.\n4.Could supercooling or mechanical impacts during transportation spuriously trigger or suppress the structural color response, thereby misreporting cold-chain status?\n5.What are the cooling and reheating rates used in the experiments, and do these rates affect reversibility or cycling stability?\n6.For several key data, please include additional replicates and provide statistical significance analyses in the Supplementary Information.\n7.Some figures/table lack sufficient detail. Please improve captions and include the necessary experimental conditions and parameters for clarity and reproducibility.", "reviewer_2": "This paper presents a development of the fabrication of Janus droplets with tunable total internal reflection and interference structural colors that respond to both surfactant composition and temperature by tailoring surfactant formulation to adjust the droplet morphology and interface curvature enabling reversible modulation of structural color appearance, including both iridescent and non-iridescent modes depending on incident light conditions.\nThis study is expected to provide a useful insight into the responsive optical material and this may be recommended for publication if the authors can successfully respond to the following comments,\n1) Conventionally, the generation of structural color requires highly ordered nano/micro structures. Top-down approaches such as photolithography allow precise fabrication while demand costly instrumentation and complex procedures. Bottom-up self-assembly, while more scalable, often suffers from difficulties in synthesizing uniform building blocks and achieving large-area structural order. These challenges significantly hinder the practical deployment of structural color materials. Recently, an alternative strategy has emerged, employing total internal reflection and interference at concave liquid–liquid interfaces within biphasic emulsion droplets to generate structural color. However, the alternative method using liquid environment also has limitation compared with the conventional top-down approach.\n2) Most of the material properties will be influenced by various environment condition. The structural color can be dynamically modulated by either surfactant composition or temperature, enabling reversible color switching via surfactant-induced morphological reconfiguration of droplets and temperature-triggered phase transitions of droplets. By integrating these dual-responsiveness properties, we demonstrate a real-time cold-chain temperature monitoring label tailored for freeze-sensitive pharmaceuticals. There can be some other aspect besides the surfactant composition or temperature affecting the optical property of the developed droplet even though the degree will be very different. Some discussion on other conditions.\n3) By integrating these dual-responsiveness properties, we demonstrate a real-time cold-chain temperature monitoring label tailored for freeze-sensitive pharmaceuticals. This monitoring system provides dual-range temperature detection, exhibiting reversible color changes near ~ 4 °C and irreversible color loss upon exposure to sub-zero temperatures (< 0 °C), attributed to phase-transition-induced disruption of internal reflection pathways. What is the most important parameter deciding the color transition? What is the temperature range for tuning? How can it be precisely tuned?\n4) When being illuminated, incident light entering the droplet can undergo total internal reflection at the curved hexadecane/HFE-7100 interface if the angle of incidence exceeds the critical angle of θ = 65.4° from arcsin(nHFE-7100/nhexadecane) (Fig. 1e). Due to the curved geometry, light rays reflect multiple times at the interface, and their path-length differences generate interference patterns localized near the three-phase contact line (hexadecane/HFE-7100/water), resulting in vivid TIRI structural colors. I wonder if this is applicable only to the specific combination of material or can be extended to general materials.\n5) In nature, after millions of years of evolution, structural colors are widespread in plants and animals, playing an important role in their predation, mating, and camouflage, due to their vividness and durability. Unlike chemical pigments, which rely on selective absorption or emission of light, structural colors are physically generated and are thus known for their brightness, environmental friendliness, high chromatic purity, and excellent resistance to fading. Recent progresses in the nature inspired dynamic color change (Nat. Rev. Bioeng., 3, 579 (2025)) needs to be briefly discussed in the introduction part. Especially, recent progresses in the thermally triggered reversible color change for soft robot applications (Nat. Comm., 12, 4658, (2021); Adv. Funct. Mater., 28, 1801847, (2018)) and strain induced reversible color change (Nat. Mater., 21, 1014 (2022) ;Nat. Mater., 21, 997 (2022)) should be briefly discussed to provide the readers with the recent related progresses.\n6) There are many other temperature dependent color changing materials. The droplets also display rapid and reversible thermal responsiveness (Fig. 5h-n). Droplets with diameters of ~ 61.2 μm show vivid pink structural color at 25 °C (Fig. 5i–j), which disappears upon cooling to 4 °C (Fig. 5k–l). This change arises from the phase transition of liquid hexadecane into a polycrystalline solid below its freezing point (18 °C), which decreases droplet transparency and volume. Some comparison on the previous temperature triggered color changing material with the current development needs to be provided.\n7) For the fabrication of the Janus droplets, the microfluidic device was consisted of two tapered cylindrical glass capillaries coaxially aligned within a square capillary. The chloroform-based oil solution was injected through the injection capillary as the inner phase, while the mixed aqueous surfactant solution was simultaneously injected from the same direction through the annular space between the injection and square capillaries, serving as the continuous phase. How is the production rate and how is the uniformity of the produced Janus droplets?\n8) The droplet may collapse for the prolonged use. How is the long term stability?\n9) The general usage of language including typos and grammatical errors need to be checked again.\n10) Some of the digital pictures are missing scale bars. They should be added to the pictures and also to the video clips.", "reviewer_3": "In this work, the authors reported Janus droplets with tunable TIRI structural colors. By tailoring surfactant formulation, the droplet morphology and interface curvature can be adjusted, enabling reversible modulation of structural color appearance. Furthermore, the structural color can be reversibly or irreversibly switched on/off in response to temperature changes, due to phase transitions that alter light reflection at the concave interface. Owing to their temperature-dependent optical response and real-time visual signaling, these droplets can serve as single-use security labels for potentially monitoring the freeze history of freeze-sensitive pharmaceuticals. The authors are suggested to address the following issues before further recommendations for publication in Nature Communications:\n1. The authors claim that “Bottom-up self-assembly, while more scalable, often suffers from difficulties in synthesizing uniform building blocks and achieving large-area structural order. These challenges significantly hinder the practical deployment of structural color materials”. Actually, there are many strategies for large-scale fabrication of structural-color materials, and some of them have been commercialized. Thus, the authors should optimize this part.\n2. As shown in Figure 1g, the structural colors are different from conventional structural-color materials. The authors should clarify the reason.\n3. What’s the relationship between the droplet size and structural color? Could the authors fabricate all colors in the visible light band?\n4. It will be fascinating if the authors can provide a theoretical model to clearly and systematically reveal the underly mechanism.\n5. How about the surfactant- and temperature-responsive stability after more than 10 cycles?\n6. As shown in Fig.6, structural-color materials have been reported for freeze-sensitive sensors in previous works. The authors should clarify their unique advantages."}, "author_response": "A point-by-point response to the editor and reviewers:\nReviewer #1:\nThe manuscript presents a Janus biphasic droplet platform that generates structural color via TIRI at\nconcave liquid–liquid interfaces. High-purity structural colors are achieved without solid patterning,\nand the system exhibits dual-mode responsiveness to surfactant composition and temperature. Overall,\nthe concept is novel and broadly applicable. Thus, I recommend its publication as a full paper after\naddressing the following issues:\nResponse: We were pleased that the reviewer found the high quality of our manuscript. In this revised\nmanuscript, we have addressed the comments and revised the manuscript accordingly.\n1. Compared with traditional cold-chain indicators, what advantages does a structural-color readout\noffer, and what are the key translation challenges? In addition, relative to other structural-color-based\ncold-chain indicators, what specific advantages does the present work provide?\nResponse: Thanks for the questions.\n(1) Compared with traditional cold-chain indicators, structural color readout offers the following\nadvantages:\na. High saturation, high brightness, and strong contrast\nTraditional cold-chain indicators tend to exhibit low-purity hues or low contrast, whereas structural\ncolors—arising from the optical interference and diffraction effects of micro/nanostructures—\nintrinsically feature high saturation, high brightness, and strong contrast.\nb. Excellent stability\nTraditional cold-chain indicators rely on chemical reactions for color change, being prone to color\ndeviation or fading with compromised long-term storage stability; whereas the coloration of\nstructural colors is based on physical interactions, which fundamentally eliminates chemical\ndegradation and thus exhibits superior storage durability and environmental stability.\nc. High responsivity to temperature variation\nTraditional cold-chain indicators predominantly rely on chemical reactions and biological activity,\nfeaturing poor precision of temperature thresholds and susceptibility to interference from\nenvironmental factors such as humidity and light. Whereas structural color cold-chain indicators\nshow high responsivity to temperature, showing rapid color switching by means of optical path\nvariation, interface morphology regulation, and droplet transparency/phase transition.\nd. Power-free and low-cost\nTraditional indicators/recorders rely on batteries and are relatively costly, while the structural color\nlabels require no power and have low material costs, allowing for large-scale application on\nindividual packages, similar to printed materials.\n(2) Compared to other structural color-based cold chain indicators, the advantages of this study include:\na. Facile preparation and low cost\nExisting structural color-based cold-chain technologies generally rely on mechanisms such as pitch\nvariation in cholesteric liquid crystal, periodic structure regulation in photonic crystal lattice, and\nreversible/irreversible nanostructure disruption. Such technologies often require expensive\nmaterials or complex preparation processes; whereas this study generates structural colors\ncompletely based on total internal reflection interference (TIRI) via liquid–liquid interface\ncurvature regulation, eliminating the need for nanofabrication or periodically arranged structures—\nwith a single microsphere sufficient to produce structural color.\nb. Combination of reversible and irreversible detection\nTraditional structural color indicators typically feature only a single response mode (either\nreversible or irreversible), whereas this study achieves dual temperature detection ranges—\nreversible on/off response at 4 ℃ (dedicated to monitoring temporary cooling processes) and\nirreversible discoloration effect below 0 ℃ (designed for recording freezing incidents). Such\ntemperature indicators that simultaneously cover dual temperature detection ranges are in high\ndemand in the industry yet rarely achievable, fulfilling the urgent need for more flexible and\npractical temperature monitoring solutions.\nc. Iridescent and non-iridescent coloration\nThe structural colors generated by TIRI exhibit vivid hues and high saturation. Their color\nperformance presents non-iridescent or iridescent characteristics that are dependent on the\nobservation angle and incident angle, thus enabling their application in anti-counterfeiting\nauthentication or the design of exclusive optical reading schemes.\nd. Excellent stability and reusability\nThe temperature indicators developed in this study maintain responsiveness and stability over 20\nthermal cycles (Fig. 5n). Although a small number of Janus droplets underwent aggregation with\nincreasing cycle numbers, triggering a gradual attenuation in reflectance spectrum intensity\n(Supplementary Fig. 8 and 9), the reflectance spectrum intensity had diminished by less than 20%\nafter 20 cycles. Furthermore, the structural color remained largely unchanged after shaking\n(Supplementary Movie 6 and Supplementary Fig. 10) and being dropped from a height of 30 cm\n(Supplementary Movie 7 and Supplementary Fig. 11). Long-term stability tests showed that the\ntemperature indicator retained its vivid structural color after 2.5 years of storage at room\ntemperature (Supplementary Fig. 12). Moreover, its color could still be fully restored after 15 days\nof storage at 4 °C (Supplementary Fig. 13). These findings collectively confirm the temperature\nindicator 's excellent overall stability.\ne. Adjustable detection temperature range\nTraditional structural color indicators often have fixed detection ranges. The indicator developed\nin this study not only achieves reversible color changes within 0-18 °C, but also allows precise\nadjustment of the temperature response range by modifying the types and proportions of alkanes\nto alter their freezing points. This is still underworking and we will report it in a separate paper.\n(3) The cold-chain indicator developed in this study based on TIRI structural color faces low\nproduction efficiency in industrial transformation. Although structural-color droplets based on TIRI\ndo not require periodically arranged micro/nanostructures, their fabrication still relies on microfluidic\ntechnology. To advance the industrialization of this technology, it is necessary to further enhance\nproduction efficiency and reduce manufacturing costs.\n2. The authors state, “Upon reheating to 25 °C, the structural color of the label is fully restored.” Will\nthis performance degrade with an increasing number of cooling–heating cycles under practical\nconditions?\nResponse: Thanks for the question.\nWith increased number of cooling–heating cycles, the Janus droplets retained their responsiveness\nand structural stability over 20 consecutive cycles. Notably, as the cycle number further increased, the\nintensity of their reflectance spectra showed a slightly decrease. This phenomenon can be attributed to\nthe aggregation of a small fraction of Janus droplets, which contributed to the progressive attenuation\nof reflectance spectral intensity (Supplementary Fig. 8). Nevertheless, after 20 cycles, the reflectance\nspectral intensity slightly decreased by less than 20% (Supplementary Fig. 9), which is sufficient to\nmeet the temperature monitoring requirements during the entire lifecycle of freeze-sensitive\npharmaceuticals.\nSupplementary Fig. 8 Optical microscopy images of the Janus droplets subjected to 20 temperature\nswitching cycles. The droplet size is 44.1µm. The Janus droplets retained their responsiveness and\nstructural color over 20 consecutive cycles.\nSupplementary Fig. 9 Reflectance spectra of the Janus droplets in the 20 cycles of temperature\nswitching.\n3. For the reported “irreversible color loss upon exposure to sub-zero temperatures (< 0 °C),” does the\nirreversibility also depend on storage duration at sub-zero temperatures? Please provide supporting\ndata.\nResponse: Thanks for the suggestion.\nIrreversibility of the structural color in the Janus droplets stored below 0 °C is independent of\nstorage duration. The key mechanism for color loss is the structural damage induced by ice crystal\nformation, rather than the time of low temperature exposure. Furthermore, long-term stability tests\nshow that the structural color can be fully restored even after 15 days of storage at 4 °C (Supplementary\nFig. 13).\nSupplementary Fig. 13 Microscopic images and reflectance spectra showing color recovery after 15\ndays of storage at 4 °C.\n4. Could supercooling or mechanical impacts during transportation spuriously trigger or suppress the\nstructural color response, thereby misreporting cold-chain status?\nResponse: Thanks for the question.\nThe core mechanism of the temperature response involves ice crystal formation in the aqueous\nphase, which disrupts the HFE-7100/water interface, followed by aggregation of the HFE-7100 phase\nduring melting, ultimately resulting in the loss of structural color. Importantly, within this system, the\nJanus droplets suspended in the aqueous phase serve as efficient ice-nucleation sites, acting like\nheterogeneous nucleation impurities that significantly suppress supercooling.\nIn terms of mechanical stability, the droplets are sealed in vial caps. The confined environment\nprovides a buffering effect that effectively minimizes the influence of shaking and impact on the\ndroplets—a feature particularly important for ensuring the reliability of temperature-indicator labels\nin practical applications. Experimentally, the structural color of the Janus microspheres remained\nlargely unchanged after gentle shaking (Supplementary Video 6 and Supplementary Fig. 10) and after\nbeing dropped from 30 cm height (Supplementary Video 6 and Supplementary Fig. 11).\nSupplementary Fig. 10 Optical microscopy images of the Janus droplets before and after gentle\nshaking.\nSupplementary Fig. 11 Optical microscopy images of the Janus droplets before and after being\ndropped from a height of 30 cm.\n5. What are the cooling and reheating rates used in the experiments, and do these rates affect\nreversibility or cycling stability?\nResponse: Thanks for the question.\nThe cooling and heating rates employed for Supplementary movie 1 and 2 were 1.2 °C/min and\n9.4 °C/min respectively. Subsequent validation using multiple rates (Supplementary Movie 3 and 4),\nincluding rapid cooling at 5.8 °C/min and slow heating at 2.6 °C/min, consistently demonstrated that\nthe system's stability and responsiveness remained unaffected by variations in heating or cooling rates.\n6. For several key data, please include additional replicates and provide statistical significance analyses\nin the Supplementary Information.\nResponse: Thanks for the suggestion.\nRegarding the surfactant cycling and temperature cycling experiments involving Janus droplets\nin the paper, we have expanded the number of cycling tests and supplemented the dataset with\ncorresponding reflection spectra collected over multiple cycles. As shown in Fig. 5, we counted the\nspectral peaks from 20 cycles of surfactant cycling and temperature cycling, with mean values of 485.0\n± 1.2 nm (Fig. 5g) and 485.9 ± 1.2 nm (Fig. 5n), respectively.\nFig. 5 Surfactant- and temperature-responsive switching of TIRI structural color in Janus droplets. (a)\nSchematic diagram showing the reversible transition of droplet structure in response to surfactant. (b-e) Reflection\noptical micrographs showing structural color fading and recovery as FS-30 and SDS are sequentially added. (f)\nReflectance spectra of the Janus droplets before/after addition of surfactants. (g) Structural color reversibility over\n20 cycles of surfactant switching. (h) Schematic of temperature-induced phase transition affecting internal reflection.\n(i,j) Photographs and reflection optical microscopy images showing the blue droplets and the blue butterfly pattern\nmade by the droplets at 25 °C. (k,l) Photographs and reflection optical microscopy images showing the colorless\ndroplets and the faded butterfly pattern at 4 °C. (m) Reflectance spectra of the droplets at different temperatures. (n)\nReversible switching performance over 20 heating–cooling cycles, confirming thermal robustness.\n7. Some figures/table lack sufficient detail. Please improve captions and include the necessary\nexperimental conditions and parameters for clarity and reproducibility.\nResponse: Thanks for the suggestion.\nTo respond, we have revised the figure captions and added the necessary experimental conditions\nand parameters in the revised manuscript.\nReviewer #2 (Remarks to the Author):\nThis paper presents a development of the fabrication of Janus droplets with tunable total internal\nreflection and interference structural colors that respond to both surfactant composition and\ntemperature by tailoring surfactant formulation to adjust the droplet morphology and interface\ncurvature enabling reversible modulation of structural color appearance, including both iridescent and\nnon-iridescent modes depending on incident light conditions. This study is expected to provide a useful\ninsight into the responsive optical material and this may be recommended for publication if the authors\ncan successfully respond to the following comments.\nResponse: We are pleased that the reviewer found the high quality of our manuscript and thank the\nreviewer for the helpful comments for further improving the manuscript.\n1. Conventionally, the generation of structural color requires highly ordered nano/micro structures.\nTop-down approaches such as photolithography allow precise fabrication while demand costly\ninstrumentation and complex procedures. Bottom-up self-assembly, while more scalable, often suffers\nfrom difficulties in synthesizing uniform building blocks and achieving large-area structural order. These\nchallenges significantly hinder the practical deployment of structural color materials. Recently,\nan alternative strategy has emerged, employing total internal reflection and interference at concave\nliquid–liquid interfaces within biphasic emulsion droplets to generate structural color. However, the\nalternative method using liquid environment also has limitation compared with the conventional top-\ndown approach.\nResponse: Thanks for the valuable comments, and we have revised the relevant sections of the\nmanuscript accordingly.\nWhile alternative methods based on liquid environments present certain limitations, they also\nopen up more possibilities for material applications. To address these challenges, various strategies\nhave been proposed. For instance, Cheng et al. (Innovation-Amsterdam 2, 100081 (2021)) encapsulated\nliquid crystal droplets within a PVA film, enabling not only tunable structural colors through\nenvironmental temperature regulation but also simultaneous shape transformation. Guan et al. (Chem\nSoc Rev 41, 3297-3317 (2012)) dispersed Fe₃O₄@PVP nanoparticles in a poly(AA-co-AM) precursor\nsolution, where an external magnetic field was applied to induce one-dimensional ordered alignment\nand generate structural color. This liquid system enabled intelligent modulation and variation of its\nstructural color. Kim et al. (Adv Mater Technol 8, 2200257 (2023)) employed microfluidic technology\nto fabricate cholesteric liquid crystals into oil-in-water-in-oil-in-water (O/W/O/W) triple-emulsion\ndroplets. Subsequently, UV light was used to crosslink and solidify their surfaces, forming photonic\nmicrocapsules with an onion-like topology. These microcapsules exhibit structural colors due to dual\nphotonic bandgaps, resulting in a rich diversity of colors on the optical palette.\nAlthough the liquid systems may have stability issues, they show rapid responsiveness to external\nstimuli, enabling their wide applications in sensors. Moreover, we found that our system was stable\nand reliable (see Supplementary Fig. 8-12). Therefore, we think our system provides an alternative\nsolution for cold-chain temperature monitoring.\n2. Most of the material properties will be influenced by various environment condition. The structural\ncolor can be dynamically modulated by either surfactant composition or temperature, enabling\nreversible color switching via surfactant-induced morphological reconfiguration of droplets and\ntemperature-triggered phase transitions of droplets. By integrating these dual-responsiveness\nproperties, we demonstrate a real-time cold-chain temperature monitoring label tailored for freeze-\nsensitive pharmaceuticals. There can be some other aspect besides the surfactant composition or\ntemperature affecting the optical property of the developed droplet even though the degree will be very\ndifferent. Some discussion on other conditions.\nResponse: Thanks for the comments.\nCurrently, there has been extensive research on the factors influencing the structural color in total\ninternal reflection (TIR). According to the light intensity formula for TIR,\n2\nm\nI = |∑ m A m (α m )[r m (α m ,\nn1)]\nexp(\n2πin1l\nm (α m ))| ,\nall possible paths n2 λ0\nthe color primarily depends on the refractive index difference between the two phases, the incident and\nexit angles, as well as the size and shape of the reflective interface (Adv Opt Mater 6, 1701009 (2018);\nAdv Mater Technol 10, 2400865 (2025); Adv Sci 12, 2413027 (2025)). Among these factors, the\nincident and exit angles, along with the geometric shape of the reflective interface, mainly influence\nthe structural color at the exit position by modulating the propagation path of light at the interface,\nthereby causing variations in the optical path difference.\nTo respond, we have added discussions in the revised manuscript.\n3. By integrating these dual-responsiveness properties, we demonstrate a real-time cold-chain\ntemperature monitoring label tailored for freeze-sensitive pharmaceuticals. This monitoring system\nprovides dual-range temperature detection, exhibiting reversible color changes near ~ 4 °C and\nirreversible color loss upon exposure to sub-zero temperatures (< 0 °C), attributed to phase-transition-\ninduced disruption of internal reflection pathways. What is the most important parameter deciding the\ncolor transition? What is the temperature range for tuning? How can it be precisely tuned?\nResponse: Thanks for the questions.\nIn our study, the color transition of the temperature-indicating label is governed by the freezing\npoints of the two phases in the Janus droplets and the freezing temperature of the surfactant solution.\nThe irreversible temperature range is below 0 °C; the reversible range spans 0-60 °C. In the range of\n0-18 °C, as temperatures fall below the freezing point of the n-hexadecane phase, the structural color\nof the Janus droplets appears colorless. In the range of 18-60 °C, vivid structural colors appear. Above\n60 °C, the Janus microsphere's two-phase structure breaks down, as it is higher than the boiling point\nof methyl nonafluorobutyl ether, causing the structural color to vanish.\nSince the structure color switching is dominated by the phase transition of alkane in the Janus\ndroplets, the transition temperature can be precisely tuned by varying the composition of the alkane\nphase. For example, when heptane/hexadecane mixture is used as the alkane phase, the transition\ntemperature can be tuned by varying the volume ratio of heptane to hexadecane. We are currently\nworking on this issue and plan to report this strategy in a following work.\nFigure R1. Photographs and reflectance spectra of the security label exposed to 25 °C → 60 °C →\n25 °C.\n4. When being illuminated, incident light entering the droplet can undergo total internal reflection at\nthe curved hexadecane/HFE-7100 interface if the angle of incidence exceeds the critical angle of θ =\n65.4° from arcsin(n-HFE-7100/n-hexadecane) (Fig. 1e). Due to the curved geometry, light rays reflect\nmultiple times at the interface, and their path-length differences generate interference patterns\nlocalized near the three-phase contact line (hexadecane/HFE-7100/water), resulting in vivid TIRI\nstructural colors. I wonder if this is applicable only to the specific combination of material or can be\nextended to general materials.\nResponse: Thanks for the comments.\nThe selection of materials for TIR structural colors is highly universal and can be extended to a\nwide range of substances. This phenomenon is governed by two fundamental conditions: first, light\nmust travel from an optically denser medium (higher refractive index) to an optically rarer one (lower\nrefractive index); second, the angle of incidence must exceed the critical angle. Any materials that\nsatisfy these two conditions can be utilized to produce vivid TIR structural colors (J Am Chem Soc 144,\n7610-7615 (2022); Chem Soc Rev 45, 6698-6724 (2016); Mater Horizons 6, 945-958 (2019)). This\nstrategy is general. In our lab, we can produce TIR structural colors in biphasic systems, like\nsilicone/polystyrene, hexadecane/polystyrene, HFE-7100/liquid crystal, and many other systems.\n5. In nature, after millions of years of evolution, structural colors are widespread in plants and animals,\nplaying an important role in their predation, mating, and camouflage, due to their vividness and\ndurability. Unlike chemical pigments, which rely on selective absorption or emission of light, structural\ncolors are physically generated and are thus known for their brightness, environmental friendliness,\nhigh chromatic purity, and excellent resistance to fading. Recent progresses in the nature inspired\ndynamic color change (Nat. Rev. Bioeng., 3, 579 (2025)) needs to be briefly discussed in the\nintroduction part. Especially, recent progresses in the thermally triggered reversible color change for\nsoft robot applications (Nat. Comm., 12, 4658, (2021); Adv. Funct. Mater., 28, 1801847, (2018)) and\nstrain induced reversible color change (Nat. Mater., 21, 1014 (2022); Nat. Mater., 21, 997 (2022)) should\nbe briefly discussed to provide the readers with the recent related progresses.\nResponse: Thanks for the suggestions.\nWith advances in the design of artificial micro- and nanostructures, the fabrication and tuning of\nstructural color materials have progressed rapidly. In particular, stimuli-responsive structural color\nmaterials that can change their colors in response to external stimuli have been prepared using smart\nmaterials (Adv Mater 30, 1706654 (2018); Adv Funct Mater 35, 2412703 (2025); Prog Mater Sci 144,\n101291 (2024); Nat Rev Bioeng 3, 579-595 (2025); Small 16, 1907626 (2020)), providing real-time\nvisible feedback to external stimuli such as temperature, magnetic field, pressure, solvent, light,\nelectric field, pH, and others (Nat Commun 14, 3960 (2023); Adv Opt Mater 11, 2203093 (2023); Sci\nAdv 7, eabh1992 (2021); Sci Adv 5, eaaw8755 (2019); Adv Funct Mater 35, 2425806 (2025); Small\nStruct 3, 2200101 (2022)). In the field of thermal-responsive color change, the integration of thermally\nsensitive structural-color materials with flexible heaters enables full-spectrum reversible color\nmodulation in soft robots, offering a novel strategy for biomimetic camouflage and human–machine\ninteraction (Prog Mater Sci 135, 101091 (2023); Opin Solid State Mat Sci 23, 13-27 (2019)). Regarding\nmechanical response, interference lithography based on photopolymerizable elastomers exploits\nstrain-induced deformation of periodic photonic nanostructures to achieve precise optical tuning\n(Mater Horizons 12, 2669-2678 (2025); Nat Commun 16, 2264 (2025)). This approach facilitates high-\nsensitivity mechanical sensing and real-time visual monitoring of deformation, meeting the demands\nof wearable devices and intelligent structural health monitoring. Owing to their immediate and\nperceptible optical response, such materials show great potential in next-generation displays, anti-\ncounterfeiting, chemical and physical sensing, and environmental monitoring (ACS Nano 17, 15180-\n15188 (2023); ACS Nano 17, 10269-10279 (2023); ACS Nano 16, 20094-20099 (2022); ACS Nano 16,\n20705-20713 (2022)). To respond, we have cited these references and added related discussion in the\nrevised manuscript.\n6. There are many other temperature dependent color changing materials. The droplets also display\nrapid and reversible thermal responsiveness (Fig. 5h-n). Droplets with diameters of ~ 61.2 μm show\nvivid pink structural color at 25 °C (Fig. 5i–j), which disappears upon cooling to 4 °C (Fig. 5k–l). This\nchange arises from the phase transition of liquid hexadecane into a polycrystalline solid below its\nfreezing point (18 °C), which decreases droplet transparency and volume. Some comparison on the\nprevious temperature triggered color changing material with the current development needs to be\nprovided.\nResponse: Thanks for the suggestion.\nCompared to other structural color-based cold chain indicators, the advantages of this study include:\na. Facile preparation and low cost\nExisting structural color-based cold-chain technologies generally rely on mechanisms such as pitch\nvariation in cholesteric liquid crystal, periodic structure regulation in photonic crystal lattice, and\nreversible/irreversible nanostructure disruption. Such technologies often require expensive\nmaterials or complex preparation processes; whereas this study generates structural colors\ncompletely based on total internal reflection interference (TIRI) via liquid–liquid interface\ncurvature regulation, eliminating the need for nanofabrication or periodically arranged structures—\nwith a single microsphere sufficient to produce structural color.\nb. Combination of reversible and irreversible detection\nTraditional structural color indicators typically feature only a single response mode (either\nreversible or irreversible), whereas this study achieves dual temperature detection ranges—\nreversible on/off response at 4 ℃ (dedicated to monitoring temporary cooling processes) and\nirreversible discoloration effect below 0 ℃ (designed for recording freezing incidents). Such\ntemperature indicators that simultaneously cover dual temperature detection ranges are in high\ndemand in the industry yet rarely achievable, fulfilling the urgent need for more flexible and\npractical temperature monitoring solutions.\nc. Iridescent and non-iridescent coloration\nThe structural colors generated by TIRI exhibit vivid hues and high saturation. Their color\nperformance presents non-iridescent or iridescent characteristics that are dependent on the\nobservation angle and incident angle, thus enabling their application in anti-counterfeiting\nauthentication or the design of exclusive optical reading schemes.\nd. Excellent stability and reusability\nThe temperature indicators developed in this study maintain responsiveness and stability over 20\nthermal cycles (Fig. 5n). Although a small number of Janus droplets underwent aggregation with\nincreasing cycle numbers, leading to a gradual attenuation in reflectance spectrum intensity\n(Supplementary Fig. 8 and 9), the reflectance spectrum intensity had diminished by less than 20%\nafter 20 cycles. Furthermore, the structural color remained largely unchanged after shaking\n(Supplementary Movie 6 and Supplementary Fig. 10) and being dropped from a height of 30 cm\n(Supplementary Movie 7 and Supplementary Fig. 11). Long-term stability tests showed that the\ntemperature indicator retained its vivid structural color after 2.5 years of storage at room\ntemperature (Supplementary Fig. 12). Moreover, its color could still be fully restored after seven\ndays of storage at 4 °C (Supplementary Fig. 13). These findings collectively confirm the\ntemperature indicator 's excellent overall stability.\ne. Adjustable detection temperature range\nCommon structural color indicators often have fixed detection ranges. The indicator developed in\nthis study not only achieves reversible color changes within 0-18 °C, but also allows precise\nadjustment of the temperature response range by modifying the types and proportions of alkanes\nto alter their freezing points. This is still underworking and we will report it in a following work.\n7. For the fabrication of the Janus droplets, the microfluidic device was consisted of two tapered\ncylindrical glass capillaries coaxially aligned within a square capillary. The chloroform-based oil\nsolution was injected through the injection capillary as the inner phase, while the mixed aqueous\nsurfactant solution was simultaneously injected from the same direction through the annular space\nbetween the injection and square capillaries, serving as the continuous phase. How is the production\nrate and how is the uniformity of the produced Janus droplets?\nResponse: Thanks for the comment and question.\nThe production rate of a single microfluidic device for Janus droplets ranges from 50 to 180\nmg/min, with the specific output determined by the flow rates of both the inner and outer phases. The\nproduction rate can be significantly increased by employing commercial microfluidic device with\nmultiple channels.\nThe prepared Janus droplets exhibit good uniformity, as detailed in Supplementary Fig. 1. We\nsuccessfully fabricated pink, green, and blue Janus microspheres with diameters of 60.5 ± 0.1 µm, 48.2\n± 0.2 µm, and 41.6 ± 0.2 µm, respectively. These droplets display vivid and uniform structural colors\nunder microscopic observation. Furthermore, Supplementary Fig. 1 d-f provides additional evidence\nof the excellent dimensional uniformity of the fabricated Janus microspheres.\n8. The droplet may collapse for the prolonged use. How is the long term stability?\nResponse: Thanks for the question.\nThe Janus droplets exhibit excellent stability. As shown in Supplementary Fig. 12, the sample was\nprepared on May 8, 2023, and after being stored in a sealed container for 2.5 years, it still displays\nbright total internal reflection (TIR) structural color, demonstrating outstanding long-term stability.\nThe droplet has a diameter of 63.1 µm and exhibits a rose-pink structural color.\nSupplementary Fig. 12 Microscopic image (with inset photograph) and reflectance spectra of the\nsample after 2.5 years of storage at room temperature. The droplets were first prepared on 8 May 2023.\n9. The general usage of language including typos and grammatical errors need to be checked again.\nResponse: Thanks for the suggestion. We have carefully checked language in the revised manuscript.\n10. Some of the digital pictures are missing scale bars. They should be added to the pictures and also\nto the video clips.\nResponse: Thanks for the suggestion.\nWe have carefully checked the scale bars of the digital pictures and videos in the revised\nmanuscript.\nReviewer #3 (Remarks to the Author):\nIn this work, the authors reported Janus droplets with tunable TIRI structural colors. By tailoring\nsurfactant formulation, the droplet morphology and interface curvature can be adjusted, enabling\nreversible modulation of structural color appearance. Furthermore, the structural color can be\nreversibly or irreversibly switched on/off in response to temperature changes, due to phase transitions\nthat alter light reflection at the concave interface. Owing to their temperature-dependent optical\nresponse and real-time visual signaling, these droplets can serve as single-use security labels for\npotentially monitoring the freeze history of freeze-sensitive pharmaceuticals. The authors are\nsuggested to address the following issues before further recommendations for publication in Nature\nCommunications:\nResponse: Thanks for the helpful suggestion and comments for further improvement of our manuscript.\nWe have revised the manuscript accordingly.\n1. The authors claim that “Bottom-up self-assembly, while more scalable, often suffers from\ndifficulties in synthesizing uniform building blocks and achieving large-area structural order. These\nchallenges significantly hinder the practical deployment of structural color materials”. Actually, there\nare many strategies for large-scale fabrication of structural-color materials, and some of them have\nbeen commercialized. Thus, the authors should optimize this part.\nResponse: We appreciate the suggestions and have revised this section accordingly.\n“Bottom-up self-assembly approach, while more scalable, remains the pivotal challenge in\nachieving high uniformity of building blocks and the efficient fabrication of large-area, long-range\nordered structures. It is worth noting that in recent years, several innovative strategies have been\ndeveloped to address these challenges, including roll-to-roll deposition technology29, shear-induced\nself-assembly30, 31, and template-directed self-assembly32. Several of these technologies have\nsuccessfully progressed toward commercialization, markedly advancing the practical deployment of\nstructural color materials.”\n2. As shown in Figure 1g, the structural colors are different from conventional structural-color\nmaterials. The authors should clarify the reason.\nResponse: Thanks for the comment.\nCompared to other conventional structural color materials, total internal reflection structural\ncolors are obtained by carefully tuning the interface curvature inside the biphasic droplet. The incident\nlight undergoes complete internal reflection at the interface and subsequently interferes near the three-\nphase contact line. As a result, the structural colors are generated and can be modulated by tailoring\nthe droplet size, eliminating the dependency on periodic array structures.\n3. What’s the relationship between the droplet size and structural color? Could the authors fabricate\nall colors in the visible light band?\nResponse: Thanks for the question.\nThe size of the Janus droplets determines the observed structural color by influencing the number\nof total internal reflections and the propagation path of light at the reflective interfaces, thereby altering\nthe optical path difference of the interfering beams at the three-phase contact line. As evidenced by\nFig. 3, we fabricated a series of Janus droplets with sizes ranging from 40.3 µm to 62.7 µm, the colors\nof which form a complete cycle from violet to red.\n4.It will be fascinating if the authors can provide a theoretical model to clearly and systematically\nreveal the underly mechanism.\nResponse: Thanks for the suggestion.\nThe generation principle and theoretical mechanism of total internal reflection structural colors\nhave formed a mature theoretical system (Adv Opt Mater 6, 1701009 (2018); Adv Sci 12, 2413027\n(2025)). When incident light travels from an optically denser medium to an optically less dense\nmedium at an angle greater than the critical angle, total internal reflection occurs at the interface\nbetween the two phases. The light eventually exits at the three-phase contact line. During this process,\ndifferences in the number of reflections among different light beams result in optical path differences.\nDue to interference at the exit point, total internal reflection structural colors are produced. According\nto the light intensity formula for total internal reflection,\n2\nm\nI = |∑ m A m (α m )[r m (α m ,\nn1)]\nexp(\n2πin1l\nm (α m ))| ,\nall possible paths n2 λ0\nthe color primarily depends on the refractive index difference between the two phases, the incident and\nexit angles, as well as the size and shape of the reflective interface (Adv Opt Mater 6, 1701009 (2018);\nAdv Mater Technol 10, 2400865 (2025); Adv Sci 12, 2413027 (2025)).\n5. How about the surfactant- and temperature-responsive stability after more than 10 cycles?\nResponse: Thanks for the question.\nWe increased the number of surfactant switching and thermal cycling. For the surfactant-\nresponsive cycle, we performed 20 switching cycles (see Supplementary Figures 6 and 7). The results\ndemonstrate that even after 20 cycles, the reflection intensity remains virtually unchanged, indicating\nexcellent repeatability and structural integrity. For the temperature-responsive cycle, the results\nindicate that Janus droplets maintain both responsiveness and stability over 20 cooling–heating cycling\ncycles. However, as the number of cooling–heating cycling cycles increased, the intensity of their\nreflectance spectra slightly decreased. A small number of Janus droplets underwent aggregation,\nleading to a gradual attenuation in reflectance spectrum intensity (Supplementary Fig. 8 and 9).\nNevertheless, after 20 cycles, the reflectance spectrum intensity had decreased by less than 20%, which\nis sufficient to meet the temperature monitoring requirements during the entire lifecycle of freeze-\nsensitive pharmaceuticals\nSupplementary Fig. 6 Optical microscopy images of the Janus droplets subjected to 20 surfactant\nswitching cycles. The Janus droplet size is 44.1µm.\nSupplementary Fig. 7 Reflectance spectra of the Janus droplets in the 20 cycles of surfactant\nswitching.\nSupplementary Fig. 8 Optical microscopy images of the Janus droplets subjected to 20 temperature\nswitching cycles. The droplet size is 44.1µm. The Janus droplets retained their responsiveness and\nstructural color over 20 consecutive cycles.\nSupplementary Fig. 9 Reflectance spectra of the Janus droplets in the 20 cycles of temperature\nswitching.\n6. As shown in Fig.6, structural-color materials have been reported for freeze-sensitive sensors in\nprevious works. The authors should clarify their unique advantages.\nResponse: Thanks for the suggestion.\nCompared to other structural color-based cold chain indicators, the advantages of this study\ninclude:\na. Facile preparation and low cost\nExisting structural color-based cold-chain technologies generally rely on mechanisms such as pitch\nvariation in cholesteric liquid crystal, periodic structure regulation in photonic crystal lattice, and\nreversible/irreversible nanostructure disruption. Such technologies often require expensive\nmaterials or complex preparation processes; whereas this study generates structural colors\ncompletely based on total internal reflection interference (TIRI) via liquid–liquid interface\ncurvature regulation, eliminating the need for nanofabrication or periodically arranged structures—\nwith a single microsphere sufficient to produce structural color.\nb. Combination of reversible and irreversible detection\nTraditional structural color indicators typically feature only a single response mode (either\nreversible or irreversible), whereas this study achieves dual temperature detection ranges—\nreversible on/off response at 4 ℃ (dedicated to monitoring temporary cooling processes) and\nirreversible discoloration effect below 0 ℃ (designed for recording freezing incidents). Such\ntemperature indicators that simultaneously cover dual temperature detection ranges are in high\ndemand in the industry yet rarely achievable, fulfilling the urgent need for more flexible and\npractical temperature monitoring solutions.\nc. Iridescent and non-iridescent coloration\nThe structural colors generated by TIRI exhibit vivid hues and high saturation. Their color\nperformance presents non-iridescent or iridescent characteristics that are dependent on the\nobservation angle and incident angle, thus enabling their application in anti-counterfeiting\nauthentication or the design of exclusive optical reading schemes.\nd. Excellent stability and reusability\nThe temperature indicators developed in this study maintain responsiveness and stability over 20\nthermal cycles (Fig. 5n). Although a small number of Janus droplets underwent aggregation with\nincreasing cycle numbers, leading to a gradual attenuation in reflectance spectrum intensity\n(Supplementary Fig. 8 and 9), the reflectance spectrum intensity had diminished by less than 20%\nafter 20 cycles. Furthermore, the structural color remained largely unchanged after shaking\n(Supplementary Movie 6 and Supplementary Fig. 10) and being dropped from a height of 30 cm\n(Supplementary Movie 7 and Supplementary Fig. 11). Long-term stability tests showed that the\ntemperature indicator retained its vivid structural color after 2.5 years of storage at room\ntemperature (Supplementary Fig. 12). Moreover, its color could still be fully restored after 15 days\nof storage at 4 °C (Supplementary Fig. 13). These findings collectively confirm the temperature\nindicator 's excellent overall stability.\ne. Adjustable detection temperature range\nCommon structural color indicators often have fixed detection ranges. The indicator developed in\nthis study not only achieves reversible color changes within 0-18 °C, but also allows precise\nadjustment of the temperature response range by modifying the types and proportions of alkanes\nto alter their freezing points. This is still underworking and we will report it in a following work."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The paper has been well improved. All of my concerns are addressed with additional discussions concerning several obscures provided. Some details are also included in the revised manuscript. I am therefore pleased to see it published as its current form.It is suggested that some of the explanations about the advantages of this research should be added to the discussion or conclusion section of the article.", "reviewer_2": "The authors responded well to the comments.", "reviewer_3": "The revision is satisfactory. Thus, I recommend this manuscript for publication in Nature Communications."}, "author_response": ""}]

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