Afterglow ice formed by phosphorescent luminophore-protein conjugates and complexes in aqueous solution at freezing temperature

Domain: chemistry
Published: 2025-12-22
Editorial outcome: major_revision

Abstract
Unlike the widely reported fluorophore-biomacromolecule systems in nucleic acid testing and immunoassay, organic afterglow luminophore-biomacromolecule conjugates and complexes remain rarely explored. Here we report the observation of organic afterglow from aqueous solutions of luminophore-protein conjugates and complexes at freezing temperature, named as afterglow ice for abbreviation. Reliable N-hydroxysuccinimide ester protocol, as well as β-diketone chemistry, are applied for protein labeling to form luminophore-protein conjugates, which exhibit intriguing afterglow at freezing temperature. Control experiments reveal that hydrophobic interaction and covalent linkage between luminophore and protein can protect organic triplet excited states from quenching. In the case of luminophore-protein complex, we observe the switching-on of organic afterglow after specific recognition of streptavidin by biotinylated luminophore, which is the example of specific recognition and sensing of biomacromolecules by organic afterglow emitters. Although it is not yet a mature technology for biomedical applications, this study represents the initial step of organic afterglow materials towards bio-labeling and bioassay fields, as well as expanding the application scenarios of bioactive products.

Introduction
Organic phosphorescent and afterglow materials, characterized by their long-lived luminescence, easily modifiable molecular structures, low toxicity, sustainability, biocompatibility, and cost-effectiveness, demonstrate significant promise for applications in biomedical imaging, information storage and anti-counterfeiting technologies, smart responsive materials and sensing detection, as well as optoelectronic devices1–15. In conventional organic systems, due to the spin-forbidden nature of singlet-to-triplet transition and weak spin-orbit coupling, the formation of triplet excited states is challenging and they are prone to deactivation at room temperature. Over the past decade, researchers have continually developed and refined design strategies for high-performance organic room-temperature phosphorescent (RTP) materials, focusing on aspects such as enhancing intersystem crossing, suppressing non-radiative deactivation, and modulating intermolecular interactions16–35. It has been reported that organic molecular systems containing heavy atoms or carbonyl groups exhibit significantly enhanced intersystem crossing efficiency and improved phosphorescent quantum yields due to strong spin-orbit coupling effects, thereby showcasing exceptional room-temperature phosphorescent performance17–23. Additionally, the introduction of rigid microenvironments to constrain molecular vibrations has emerged as an effective strategy for inhibiting non-radiative deactivation processes24–28. Techniques such as constructing rigid molecular frameworks, crystal engineering, creating glassy environments, and employing two-component doping methodologies are commonly utilized29–35. Furthermore, by modulating intermolecular interactions, such as π-π stacking, hydrogen bonding, van der Waals forces, and supramolecular complexation, researchers can protect the triplet excited state, consequently enhancing the phosphorescent emission efficiency and stability of the materials36–41. Through manipulation of excited states, a variety of organic RTP systems with excellent phosphorescent properties and diverse functionalities have been successfully developed1–15,42,43.

In addition to advanced anti-counterfeiting and information encryption applications1–15, researchers have recently leveraged the environment-sensitive characteristics of RTP materials to develop smart sensors. These materials enable the detection of external environmental changes, such as chemical stimuli, illumination, temperature, pH, moisture, and mechanical force, by monitoring variations in luminescence intensity, lifetime, or spectral properties without interference from background signals, demonstrating functionalities in chemical sensing, gas detection, water quality analysis, and environmental pollutant monitoring44–60. Literature reports have shown that supramolecular inclusion-based RTP systems exhibit selectivity towards luminescent guests; for instance, the degree of RTP enhancement in solid-state crown ether RTP systems varies upon the association with different metal ions56. Similarly, a reported study showed that organic afterglow materials dispersed in aqueous environments can diminish or quench in the presence of ferric and ferrous ions59. Despite these intriguing advancements, there have been few report of responsive RTP systems capable of potentially analyzing and detecting biomacromolecules, nor studies focused on their potential specific recognition of biomacromolecules. In contrast, in the application fields of nucleic acid testing and immunoassays, organic fluorescent molecules can effectively cooperate with precisely structured biomacromolecules, either through chemical conjugation or non-covalent binding, leveraging the specificity of biomacromolecule recognition alongside the optical reporting capabilities of organic fluorescent molecules to achieve high-value biomedical applications61,62. Given this context, we believe that the construction of conjugates and complexes between biomacromolecules and afterglow molecules will be critically important for advancing the biomedical applications of these luminescent systems, particularly given that afterglow materials exhibit a significantly longer luminescence lifetime—an attribute that is essential for high-contrast bioimaging and sensitive bioanalysis. Previous studies showed the covalently linking of bromo-substituted naphthalimide derivative to bovine serum albumin and the measurement of the RTP properties of the resulting protein-dye conjugate63. Our recent work has doped difluoroboron β-diketonate (BF2bdk) molecules into matrices such as silk cocoons, shells, and feathers to develop biomass-based afterglow materials64. These investigations have primarily examined afterglow properties in solid-state forms63,64, with no reported studies on luminophore-protein conjugates or complexes in solution or dispersion states.

Based on our research experience and literature reports, we find that the realization of afterglow emission from organic molecules in solution or dispersion states is inherently challenging due to the typically low afterglow emission rate constant for organic phosphorescent molecules (for afterglow lifetimes exceeding 100ms, the afterglow rate constant should be <10s⁻¹), in contrast to the significantly higher non-radiative deactivation and oxygen quenching rates in these environments, often reaching 10² to 10³ s⁻¹ or even greater. In biological system, there exists an enzyme named horse liver alcohol dehydrogenase whose tryptophan residue (Trp 314 in the sequence) can absorb ultraviolet light to form a triplet excited state (T1) through intersystem crossing, and the enzyme’s rigid microenvironment protects this T1 state, enabling afterglow emissions at room temperature with a phosphorescence lifetime around 0.1 s65. Inspired by this, we propose that if organic luminophore were covalently linked to or complexed with proteins, certain regions of the protein might protect the luminophore’s T1 states. Furthermore, by appropriately lowering the temperature to suppress non-radiative deactivation and oxygen quenching of T1 excited state, such a luminophore-protein conjugate or complex system may enable afterglow emissions at ambient or conventional refrigerator temperatures.

Here we select proteins with hydrophobic regions and hydrophobic luminescent molecules to construct organic luminophore-protein conjugates and complexes in aqueous phases through chemical conjugation and non-covalent binding, respectively (Fig. 1). Driven by hydrophobic interactions, the luminescent molecules would associate with the hydrophobic microregions of the proteins. In the investigation of the organic luminophore-protein conjugates, we initially synthesized luminescent molecules containing N-hydroxysuccinimide (NHS) ester groups and employed bovine serum albumin (BSA) as a model protein, utilizing the traditional NHS ester labeling method to study this system. The resulting aqueous solution of the luminophore-BSA conjugates exhibited significant organic afterglow at freezing point (that is the afterglow ice), while free luminescent molecules and several other control samples showed no notable afterglow under the same conditions. In addition, we introduced a benzophenone functional group to enhance the intersystem crossing of the luminescent molecules, leading to stronger afterglow from the luminophore-BSA conjugates at freezing point. Moreover, we developed a β-diketone-based protein labeling method that achieved bright afterglow across the full color spectrum for organic luminophore-BSA conjugates. We subsequently modified the surface of polymer microspheres with luminophore-protein conjugates to obtain afterglow protein microspheres. These microspheres inherit the properties of afterglow luminophore and proteins, thereby offering potential applications in bio-analysis and bio-detection. In our study of organic luminophore-protein complexes, we utilized the classic streptavidin (SA) and biotin system (with a binding constant on the order of 10¹⁵ M⁻¹) for proof-of-concept demonstration. The synthesized biotinylated luminophore specifically binds to SA, resulting in a complex that exhibits visible phosphorescent afterglow at freezing temperature. This indicates that the specifically modified phosphors have potential for the specific recognition of proteins. Besides, we perform photophysical studies of luminophore-protein complexes in bile acid systems and demonstrate that the afterglow ice phenomenon provides a visual pathway to identify target proteins for specific biologically active compounds.

Results
Afterglow Luminophore-Protein Conjugates Obtained by NHS Ester Chemistry
We selected BSA as a model protein due to its abundant hydrophobic microregions, which allow for reversible binding with various substances, particularly those with low water solubility66. The naphthalene group, which is hydrophobic, is a well-known phosphorescent moiety; here we chose methoxynaphthalene as phosphorescent unit. By chemical modification, we synthesized 1NAP-NHS to label BSA, subsequently obtaining an aqueous solution of 1NAP-BSA conjugates after extensive dialysis against deionized water (Fig. 2). The average molecular weight of 1NAP-BSA conjugates by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (66960) exceeds that of BSA (66531) by 429 Da (Supplementary Fig. 1), corresponding to the addition of approximately two 1NAP group. These results agree with the feeding molar ratio of 1NAP-NHS/BSA (2/1) and the estimated labeling efficiency (95.6%) (Supplementary Fig. 2). It is known that MALDI-TOF works at relatively high energy, where weak bonds and non-covalent binding would be disrupted, so the molecular increase by 429 Da can be unambiguously attributed to the covalent linkage of 1NAP group to BSA through NHS ester chemistry.

The solution of 1NAP-BSA conjugates after dialysis was stored in the freezer (-20 °C) of a refrigerator (Fig. 2a), and it was observed that the frozen 1NAP-BSA conjugates emitted yellow afterglow when excited by a handheld UV lamp at 365nm (Fig. 2c). Notably, the afterglow emission remained significant when the frozen 1NAP-BSA conjugates turned into an ice-water mixture (0 °C).

UV-Vis absorption analysis of the 1NAP-BSA conjugates after dialysis revealed an absorption band in the range of 300–350nm (Fig. 2b), which can be exclusively assigned to 1NAP group, confirming the successful labeling of BSA with 1NAP group. Additionally, photoluminescence spectroscopy conducted on the frozen 1NAP-BSA conjugates showed a delayed emission peak at 550nm under 365nm excitation (Fig. 2c), which closely matched the delayed emission spectrum of 1NAP when doped in the small-molecule organic matrix such as benzoic acid (Supplementary Fig. 3). The average phosphorescence lifetime (τP,AVE) of the frozen 1NAP-BSA conjugates was measured to be 290ms (Fig. 2d). To further elucidate the origin of phosphorescence, several control experiments were performed. Firstly, a dialyzed solution of pristine BSA was prepared following identical experimental protocols but without the addition of 1NAP-NHS, and no afterglow was observed at -20 °C upon 365nm excitation (Supplementary Fig. 4). Furthermore, both high-concentration aqueous BSA solution and BSA powder exhibited no afterglow at -20 °C under 365nm excitation, although the BSA powder displayed faint and noisy delayed emission spectra (Supplementary Fig. 5). Moreover, for 1NAP-PEG aqueous solution at 15 mg/mL (1NAP is not soluble in water, so we use polyethylene glycol (PEG) to modify 1NAP into 1NAP-PEG, Supplementary Information), we found that the frozen 1NAP-PEG solution (1NAP group, 1.86 mg/mL) show fluorescence emission but no afterglow under 365nm excitation (Supplementary Fig. 6a), contrasting sharply with the evident afterglow emitted by the frozen 1NAP-BSA conjugates at a low concentration (BSA, around 1 mg/mL; 1NAP group, about 0.024 mg/mL) (Supplementary Fig. 6b), thereby underscoring the importance of the hydrophobic microregions in proteins. Finally, upon non-covalently loading 1NAP onto BSA, the frozen solution at -20 °C yielded very weak afterglow under 365nm excitation (Supplementary Fig. 7). This experiment not only elucidates that the observed bright afterglow does not arise from a simple blending of luminescent molecules and proteins but also underscore the necessity of covalent linkage between 1NAP and BSA. Through these control experiments, we propose that, driven by hydrophobic interactions, 1NAP-NHS can bind to some hydrophobic microregion of BSA and then form conjugates with BSA via amide bonds. The synergistic effects of hydrophobic interactions and covalent bonding protect 1NAP’s triplet excited state, thereby enabling afterglow emission from the conjugates at conventional refrigerator temperatures.

Nuclear magnetic resonance (NMR) technique is used to study the mobility of luminophore in different microenvironments. When 1NAP groups are covalently linked to PEG or ε-polylysine (EPL) chains, the resultant 1NAP-PEG and 1NAP-EPL samples in D2O show sharp and well-resolved 1H NMR signals in the aromatic region (Supplementary Figs. 8, 9). The 1NAP groups have free motion in D2O to give sharp peaks, because the hydrophilic polymers (PEG and EPL) endow 1NAP groups with excellent solubility in D2O. In contrast, 1H NMR spectrum of 1NAP-BSA conjugates in D2O shows the disappearance of NMR signals of 1NAP groups in the aromatic region (Supplementary Fig. 10), which suggest the motion of 1NAP groups is seriously restricted within BSA67. The hydrophobic 1NAP groups within some hydrophobic microregion of BSA have largely restricted motion, which can significant reduce nonradiative decay of 1NAP’s triplet excited states, leading to the emergence of afterglow at freezing temperature.

Subsequently, we selected various phosphorescent moieties including acridone, pyrene, naphthalene, benzophenone, biphenyl, and fluorene for labeling BSA via NHS ester chemistry, resulting in the formation of diverse luminophore-BSA conjugates (the chemical structures of corresponding Ar-NHS compounds can be found in Fig. 2e). The labeling efficiency estimated by UV-vis technique is >95% (Supplementary Fig. 2). Among these, the Acridone-BSA conjugates displayed bright blue-green afterglow and revealed a dual emission in its delayed spectrum (Fig. 2f, g). Based on our previous studies on acridone derivatives68,69, the dual emission can be assigned as thermally activated delayed fluorescence and phosphorescence. The Pyrene-BSA conjugates demonstrated a vivid red afterglow with phosphorescence maximum at 620nm (Fig. 2h) and an average afterglow lifetime of 188ms (Supplementary Fig. 11). The frozen 2NAP-BSA conjugates exhibited strong yellow-green afterglow emission and an average afterglow lifetime of 265ms (Supplementary Fig. 12). The BP-BSA, MeOBip-BSA, and Fluorene-BSA conjugates exhibited relatively weak afterglow emissions (Supplementary Figs. 13, S14).

Bright afterglow conjugates via ArBP-NHS labeling of proteins
To enhance the afterglow performance of luminophore-BSA conjugates, we introduced a benzophenone functional (BP) group into the Ar-NHS system to form ArBP-NHS compounds (Fig. 3). Benzophenone is recognized as a significant phosphorescent emitter, characterized by an intersystem crossing efficiency that approaches unity. However, it exhibits a notably short phosphorescence lifetime, typically ranging from 0.1 to 1ms, primarily due to the pronounced n-π* transition inherent to its triplet state. Recent research indicates that advanced charge transfer technology can maintain strong intersystem crossing in benzophenone-derived organic systems and simultaneously reduce phosphorescence rate constant (kP) to small values23,70. When non-radiative decay pathways are restricted using a rigid environment, it becomes possible to achieve highly efficient and persistent room-temperature phosphorescence. The benzophenone moiety is electron-deficient. By connecting it with electron-donating aromatic groups, an intramolecular charge transfer (ICT) system is established. The donor group contributes actively to the triplet excited state formation of the resulting system. If the donating group possesses a low-energy T1 state of localized excitation (LE) character, the T1 state of the resulting molecules would consist of significant portion from the donating group’s T1 state. As a result, the resulting molecules’ T1 states would inherit the donating group’s LE character and thus have small kP. Naphthoic acid features a relatively low T1 energy level; upon conjugation with the BP group, the resultant NAPBP-COOH molecule capitalizes on the localized excitation characteristics of naphthoic acid’s triplet state, thereby facilitating the formation of a long-lived T1 state within NAPBP-COOH23,70. This molecular design effectively preserves the robust intersystem crossing capability of the benzophenone framework while attenuating the contribution of the n-π* transition to the T1 state. Consequently, this configuration endows the luminophore with both high intersystem crossing efficiency and a small phosphorescence rate constant. The efficacy of this molecular design is corroborated by experimental data from small-molecule doping systems, which demonstrate bright phosphorescence with an emission maximum/shoulder at 527/555nm and a phosphorescence lifetime extending up to 524ms (Supplementary Fig. 15). Consequently, we utilized NAPBP-NHS to label BSA, obtaining NAPBP-BSA conjugates. The UV-vis absorption spectrum of the dialyzed solution revealed the presence of an absorption band corresponding to NAPBP (Fig. 3b). The aqueous solution of the conjugates at room temperature shows negligible delayed emission signals (Supplementary Fig. 16), which can be explained by the active non-radiative decay and oxygen quenching of luminophore’s triplet excited states at fluidic aqueous solution. After storing the dialyzed solution in a refrigerator (-20 °C), the frozen NAPBP-BSA conjugates under 365nm excitation displayed phosphorescence maximum/shoulder at 526/559nm in the delayed emission spectrum (Fig. 3c). The average afterglow lifetime was measured to be 410ms (Fig. 3d). In comparison to the 2NAP-BSA conjugates (without BP attachment), the phosphorescence intensity and lifetimes are significantly enhanced in the system of NAPBP-BSA conjugates; under excitation by a 365nm handheld UV lamp, a brighter and longer afterglow emission was visually observed in NAPBP-BSA system (Fig. 3c). We collect temperature-dependent delayed emission spectra and emission decay profiles for the NAPBP-BSA conjugates. The delayed emission intensity is very weak at 278K because the active non-radiative decay and oxygen quenching of triplet excited states in the fluidic aqueous solution. Upon freezing, the delayed emission intensity has significant increase (Supplementary Fig. 17a), indicating the essential role of ice for the protection of NAPBP’s triplet excited states. It has also been found that, by further decreasing the temperature, the delayed emission intensity and phosphorescence lifetimes increase (Supplementary Fig. 17b), which can be attributed to better protection of NAPBP’s triplet excited states within the ice at lower temperature. Time-dependent density functional theory (TD-DFT) calculations indicated that the S1 excited state of NAPBP-COOH displayed typical characteristics of intramolecular charge transfer, there are rich ISC channels with substantial spin-orbit coupling matrix element (SOCME) values, and the T1 excited state exhibited features indicative of significant localized excitation (Supplementary Fig. 18). These findings suggest that the NAPBP system possesses strong ISC along with a slow phosphorescence decay rate, which is consistent with our experimental observation.

We understand that some compounds may precipitate at low temperature that may be responsible for the emergence of afterglow in the present system. To study this, aqueous solution of NAPBP-BSA conjugates are immediately frozen by liquid nitrogen and then placed in -20 °C refrigerator for 1 day. The sample shows homogenous afterglow upon removing excitation source (Supplementary Fig. 19). Besides, the NAPBP-BSA conjugates have been diluted to 1 mg/mL. The frozen solution of this diluted sample still shows significant afterglow (Supplementary Fig. 20A–C). Since the extent of precipitation or phase separation would be relatively low during rapid freezing and for samples at low concentration, these results suggest the emergence of afterglow in the present study is unlikely caused by precipitation or phase separation. To further study whether protein precipitation/phase separation during freezing contributes to the emergence of afterglow, we covalently link PEG chains to NAPBP-BSA conjugates; the steric repulsion between PEG chains would minimize the possibility of protein aggregation especially in the case when the NAPBP-BSA conjugates are heavily decorated with PEG chains. After being frozen, the PEG-decorated NAPBP-BSA conjugates have been found to show similar afterglow properties compared to NAPBP-BSA conjugates (control sample, without PEG) (Supplementary Fig. 20D–Q). These studies show that protein aggregation may occur during freezing in the case of luminophore-protein conjugates but has insignificant contribution to afterglow emergence in the present system, and thus support our proposed mechanism of synergistic effects of conjugate formation and hydrophobic interactions at freezing temperature for the emergence of afterglow.

It should be noted that, in the NAPBP-BSA system, the T1 states of localized excitation character with very small oscillator strength would have limited tendency to undergo photochemical reaction with protein. Besides, NAPBP-BSA conjugates at low concentrations (2.5 mg/mL to 7.5 mg/mL) have negligible or low cytotoxicity to NIH3T3 cells (Supplementary Fig. 21). The cytotoxicity increases with sample concentration but is still acceptable at higher concentrations (10 mg/mL, Supplementary Fig. 21).

By substituting the NAP group with other functional groups, such as dimethylfluorene, pyrene, benzophenone, biphenyl, spirobifluorene, and triphenylamine, we synthesized six additional ArBP-NHS molecules (Fig. 3e). After BSA labeling, all six ArBP-BSA conjugates exhibited persistent afterglow emission at freezing temperature. Notably, under 365nm excitation, four of the systems demonstrated afterglow lifetimes exceeding 300ms (Supplementary Figs. 22, S23). The FluoreneBP-BSA conjugates emitted a bright bluish-green afterglow (Fig. 3f), with an average phosphorescence lifetime of 511ms (Supplementary Fig. 22a), while the PyreneBP-BSA conjugates emitted a vivid red afterglow at 615nm (Fig. 3g), with an average phosphorescence lifetime of 339ms (Supplementary Fig. 22b). In the examples of NAPBP-BSA, PyreneBP and MeOBipBP, the introduction of the BP group significantly enhanced the afterglow of the luminophore-BSA conjugates, when compared to those in 2NAP-BSA, Pyrene-BSA and MeOBip-BSA systems (Fig. 3c, g, i). These results further validate the effectiveness of the ArBP-NHS molecular design. Photophysical data for other ArBP-BSA conjugates are detailed in Fig. 3h, Supplementary Figs. S23, S24.

Interesting β-Diketone-based covalent labeling of proteins: a serendipitous discovery
Building upon the above NHS ester chemistry, we aimed to label BSA with our phosphorescent difluoroboron β-diketonate (BF2bdk) molecules such as 2COOHNAPBF2-NHS, hypothesizing that this would yield robust persistent luminescence (Fig. 4a). The results demonstrated that the obtained luminophore-protein conjugates at freezing point emitted an expected bright yellow-green long afterglow. Surprisingly, control experiments revealed that treatment of 2COOHNAPBF2 (without NHS ester) with BSA under identical conditions also resulted in intense yellow-green afterglow emission (Fig. 4b). Initially, we speculated that this phenomenon arises from a strong non-covalent interaction between 2COOHNAPBF2 and BSA; the combination of hydrogen bonding and hydrophobic interaction between 2COOHNAPBF2 and BSA might be responsible for this strong non-covalent interaction. In control experiment, we removed the carboxyl group of 2COOHNAPBF2 to form 0COOHNAPBF2, and subjected it to the same experimental procedures (stirring with BSA, dialysis, and being stored at -20 °C). We still observed bright afterglow emission, thus ruling out the contribution of carboxyl group/hydrogen bonding for the emergence of afterglow. We then proposed an alternative mechanism whereby, in alkaline environments, the BF2 group of the phosphorescent molecule readily dissociates. The resulting β-diketone moiety can react with the amine groups of proteins to form β-enaminone compounds (Fig. 4c)71,72, suggesting that the afterglow in this system is induced by the covalent linkage between β-diketone and BSA. The ice matrix also plays essential role for the emergence of afterglow as evidenced by the temperature-dependent delayed emission spectra and emission decay profiles, where the phosphorescence intensity and lifetimes increase upon freezing and lowering temperature (Supplementary Fig. 25). In control experiment, the aqueous solution of the conjugates at room temperature has negligible delayed emission signals because of the active non-radiative decay and oxygen quenching in fluidic aqueous medium (Supplementary Fig. 26). Besides temperature, we also investigate the contribution of hydrophobic effect on the afterglow property. Sodium dodecyl sulfonate (SDS) is known to disrupt hydrophobic interactions in protein systems. We add SDS into the aqueous solution of 2COOHNAP-diketone-BSA conjugates. After being stored at -20 °C, the frozen sample with the presence of SDS shows much weaker phosphorescence than the frozen sample without SDS (Supplementary Fig. 27). This observation supports our proposed mechanism that hydrophobic interaction and covalent linkage between luminophore and protein can protect organic triplet excited states from quenching.

To validate this proposed β-diketone-based covalent labeling chemistry, we divided 2COOHNAPBF2 into five groups under alkaline conditions: one untreated group and the remaining groups treated with various amines, including glycine (Gly), L-lysine (Lys), L-arginine (L-Arg), and n-butylamine. High-performance liquid chromatography with mass spectrometry (HPLC-MS) was employed to monitor the formation of β-diketone and β-enaminone products. The HPLC-MS results indicated that free 2COOHNAPBF2 generates a β-diketone with a m/z of 287 in mildly alkaline conditions (Supplementary Fig. 28). Additionally, β-diketone peaks and corresponding β-enaminone peaks were observed for all four amine-treated systems (Supplementary Figs. 29, S30), confirming that β-diketones can react with amine groups to produce β-enaminones. Moreover, we aimed to further establish the feasibility of β-diketone-based protein labeling through SDS-PAGE electrophoresis. Following reactions of five BF2bdk compounds with BSA in alkaline conditions and subsequent dialysis, we obtained five BF2bdk-BSA aqueous solutions exhibiting fluorescence when excited at 365nm. These were tested alongside a control group of pristine BSA in SDS-PAGE. All six samples exhibited similar migration patterns under daylight. Under 365nm excitation, the BF2bdk-BSA solutions showed fluorescence, while the pristine BSA didn’t exhibit any fluorescence (Fig. 4d and Supplementary Fig. 31). This result corroborates the ability of β-diketones to achieve covalent labeling of proteins.

Consequently, we aim to develop a β-diketone-based protein labeling strategy that triggers organic afterglow in luminophore-protein conjugates. As illustrated in Fig. 4c, we synthesized the 2COOHNAP-diketone molecule and labeled it with BSA. UV-Vis absorption spectral data from the dialyzed solution of 2COOHNAP-diketone-BSA confirmed successful labeling of BSA by the β-diketone (Fig. 4e). Upon refrigeration, the dialyzed solution emitted a bright yellow-green afterglow when excited by a 365nm handheld UV lamp, and its delayed emission spectrum revealed a distinct phosphorescence band of 2COOHNAP-diketone (Fig. 4f), with an average afterglow lifetime of 201ms (Fig. 4g), which is supported by experimental data from small-molecule doping systems (Supplementary Fig. 32). Furthermore, we conducted a control experiment without the presence of BSA, modifying 2COOHNAP-diketone with hydrophilic polymer PEG and preparing an aqueous solution at 13 mg/mL (2COOHNAP group, about 2.02 mg/mL), which was subsequently frozen in the refrigerator; under irradiation with a 365nm UV lamp, no afterglow emission was detected (Supplementary Fig. 33). In contrast, the 2COOHNAP-diketone-BSA conjugates exhibited significant afterglow behavior even at 1 mg/mL (2COOHNAP group, about 0.012 mg/mL), underscoring the importance of BSA’s hydrophobic microregion for the emergence of afterglow. Integrating the results that the frozen solution of single-component BSA showed no afterglow (Supplementary Fig. 4), we propose that 2COOHNAP-diketone interacts with some hydrophobic microregion of BSA through hydrophobic interactions. This interaction facilitates the formation of luminophore-protein conjugates via reactions between the β-diketone and amino groups of the protein. As a result of the synergistic effect of hydrophobic interactions and covalent bonding, the triplet excited state of the luminophore is protected, enabling phosphorescent emission of the conjugates at freezing temperatures. The 2COOHNAP-diketone-BSA conjugates show negligible or low cytotoxicity to NIH3T3 cells at low concentrations (2.5 mg/mL to 7.5 mg/mL) and acceptable cytotoxicity at a higher concentration (10 mg/mL, Supplementary Fig. 34).

Given that the BF2bdk molecules can readily convert into β-diketone compounds in alkaline condition, more BF2bdk molecules with diverse structures have been synthesized via our cascade reaction (Fig. 5) for covalent labeling of BSA. All 19 BF2bdk molecules can successfully label BSA, resulting in conjugates that exhibited full-spectrum afterglow emission at freezing point. By UV-vis measurement, the labeling efficiency can be estimated to be 23.1% to 89.5% (Supplementary Fig. 35). Notably, upon excitation at 365nm, the BipCOOHBF2-BSA conjugates (Fig. 6a), OMeIndBF2-BSA conjugates (Fig. 6b), IndBF2-BSA conjugates (Fig. 6c), BrTPABF2-BSA conjugates (Fig. 6d), 1COOHNAPBF2-BSA conjugates (Fig. 6e), and PyreneBF2-BSA conjugates (Fig. 6f) demonstrated bright blue, cyan, green, yellow-green, yellow, and red phosphorescence, respectively, with wavelengths ranging from 477 to 630nm. The emission decay curve corresponding to the six BF2bdk-BSA conjugates are presented in Supplementary Fig. 36. Moreover, the steady-state and delayed spectra of the BrTPABF2-BSA and TPABF2-BSA conjugates overlapped almost completely, resulting in afterglow emission based on the TADF mechanism (Fig. 6d and Supplementary Fig. S37). The CorBF2-BSA conjugates exhibited a long phosphorescence lifetime of up to 600ms (Supplementary Fig. 38). We apply deuteration technique to increase phosphorescence lifetimes of luminophore-protein conjugates. C-D stretching vibration is much weaker than C-H stretching vibration. Deuteration of luminophores can reduce the energy of effective non-classical vibrational mode involved in the nonradiative transition, decrease Franck-Condon factor, and consequently reduce nonradiative decay, leading to the elongation of phosphorescence lifetime24. Deuterated CorBF2 (DCorBF2) has been prepared and used to label BSA; the synthetic details can be found in Supplementary Information. The resultant aqueous solution DCorBF2-BSA conjugates at frozen state display phosphorescence lifetime of 2.29 s (Fig. 6g, h), which remains very rare in the related afterglow material systems (Supplementary Fig. 39). Supplementary Fig. 39 summarizes the reported afterglow materials in aqueous systems including supramolecular assemblies, suspension, emulsion and hydrogels, from which one can find that it is challenging to prepare materials with long phosphorescence lifetimes because of the active non-radiative decay and oxygen quenching in aqueous systems. Compared to these reported studies, the phosphorescence lifetimes of our DCorBF2-BSA conjugates at frozen state are among the longest in the reported studies of aqueous afterglow systems. The photophysical data for the other BF2bdk-BSA conjugates are detailed in Figs. S40–S48.

Furthermore, by varying the types of proteins and using 2COOHNAPBF2 as the luminophore, we successfully labeled streptavidin (SA), γ-globulin, silk protein, concanavalin A, and insulin, resulting in five conjugates that emitted afterglow visible by the human eye at freezing temperatures (Fig. 7). The emission decay curve corresponding to the five 2COOHNAPBF2-protein conjugates are presented in Supplementary Fig. 49. Among these conjugates, the 2COOHNAPBF2-insulin conjugates exhibited the brightest yellow-green phosphorescent afterglow (Fig. 7f). These results further support the versatility of β-diketone-based method for protein labeling and its ability to trigger phosphorescent emission in the resulting conjugates.

For the β-diketone-based method, the luminophors (either the β-diketone or the BF2bdk) are relatively stable and allow long-term storage under ambient conditions. Besides, our research group builds a molecular library that now contains over 500 BF2bdk compounds, from which we can choose luminophors with diverse phosphorescence colors and other desired properties. Moreover, most of the luminophores have intramolecular charge transfer property, which can be readily excited by UVA and visible lights, show strong intersystem crossing efficiency and modest phosphorescence decay rate. Consequently, the resultant luminophore-protein conjugates would exhibit significant phosphorescence property at freezing temperature.

Organic afterglow protein microspheres
Given the promising applications of luminescent microspheres in the analysis and detection of biomacromolecules73–76, we conducted a study on the modification of polymer microspheres with luminophore-protein conjugates (Fig. 8a). Specifically, we selected monodisperse carboxylated polystyrene microspheres with a size of 5 micrometers. Initially, the microspheres were coated with positively charged polyethyleneimine to impart a positive surface charge. Subsequently, negatively charged 2COOHNAPBF2-BSA conjugates were electrostatically attracted to and anchored onto the surface of the microspheres. Under 365nm UV lamp, the decorated microspheres emitted bright afterglow visible by the human eye, as collaborated by delayed emission spectra (Supplementary Fig. 50a). Scanning electron microscopy (SEM) analysis revealed that the decorated microspheres possessed a regular morphology and uniform size distribution (Fig. 8b). By replacing polyethyleneimine by a more biocompatible polymer, poly-L-arginine, we also successfully prepared 2COOHNAPBF2-BSA-decorated microspheres, which maintained excellent afterglow properties and consistent microsphere morphology (Fig. 8c), as collaborated by delayed emission spectra (Supplementary Fig. 50b). In control experiments, pristine BSA was used in the place of 2COOHNAPBF2-BSA, the resultant microspheres retained good morphology but exhibited no afterglow (Supplementary Fig. 51). These experiments demonstrated that the observed phosphorescence in the afterglow protein microspheres originated from the luminophore-BSA conjugates. In addition to the polyelectrolyte coating method, we activated the carboxylated polystyrene microspheres with NHS ester and covalently linked the bright phosphorescent 2COOHNAPBF2-insulin conjugates to the microsphere surface (Fig. 8a). The resulting afterglow protein microspheres emitted bright yellow-green phosphorescence when excited with a handheld 365nm UV lamp (Fig. 8d). These microspheres inherit the properties of organic luminophore and proteins, suggesting their potential for analyzing and detecting biomacromolecules.

Afterglow luminophore-Protein Complex via Specific Recognition
We further investigated the properties of luminophore-protein complex systems. We selected the classic streptavidin-biotin (SA-Biotin) pair, renowned for its binding constant on the order of 10¹⁵ M⁻¹77,78, to provide specific recognition interaction in the present system. For the molecular design, NAPpBP was chosen as the phosphorescent moiety because of its strong ISC and potential of long-lifetime phosphorescence. We first synthesized NAPpBP-NH2 and then connected it with biotin to obtain NAPpBP-Biotin (Fig. 9a). The corresponding phosphorescent molecule NAPpBP-NHCOCH3 has also been synthesized, which exhibited a bright yellow afterglow when doped in suitable small-molecule matrix and excited at 365nm (Supplementary Fig. 52). According to the reported studies79,80, chemical linkage of functional moiety to biotin’s carboxylic acid group would not affect the specific binding property between biotin group and SA.

For the formation of luminophore-protein complex (Fig. 9b), NAPpBP-Biotin was added into SA aqueous solution and the obtained mixture was incubated at room temperature for 2h. After dialysis against water, the resultant solution has been found to show UV-vis absorption band in the region of 300–400nm, which can be exclusively assigned to NAPpBP group (Fig. 9c). At freezing point, the solution has been found to emit a yellow afterglow upon 365nm excitation. Its delayed emission spectrum revealed a phosphorescence band at 537nm (Fig. 9d), similar to that of NAPpBP-NHCOCH3 sample in doped state (Supplementary Fig. 52). In control experiments, when NAPpBP-Biotin was not added, the frozen solution of pure SA didn’t show afterglow (Supplementary Fig. 53). The frozen solution of Biotin-SA also didn’t show afterglow (Supplementary Fig. 54). NAPpBP was also modified with PEG and the aqueous solution of NAPpBP-PEG at 10 mg/mL (NAPpBP group, about 1.49 mg/mL) at frozen state showed no afterglow (Supplementary Fig. 55). In contrast, a clear afterglow was observed for the NAPpBP-Biotin-SA complex at 1 mg/mL (NAPpBP group, about 0.088 mg/mL), confirming the significance of the protein’s hydrophobic microregion for afterglow emergence. Furthermore, when we replaced NAPpBP-Biotin with NAPpBP-NHCOCH3, and subjected it to the same experimental conditions with SA, no afterglow was produced (Supplementary Fig. 56). Similarly, treatment of NAPpBP-Biotin with BSA under identical conditions resulted in only a very weak afterglow (Supplementary Fig. 57). These experiments demonstrate that NAPpBP-Biotin can specifically bind to SA, forming the NAPpBP-Biotin-SA complex. Within this complex, the T1 state of NAPpBP moiety upon UV excitation can be effectively protected by the hydrophobic microregion of SA, allowing for the bright afterglow emission. The NAPpBP-Biotin-SA complex (0.5 mg/mL to 2.0 mg/mL) has negligible cytotoxicity to NIH3T3 cells (Supplementary Fig. 58). Moreover, we also synthesized 1NAP-Biotin to complex with SA under the same experimental conditions; the 1NAP group has weak phosphorescent property compared to NAPpBP. The resultant 1NAP-Biotin-SA complex in the frozen state yielded no afterglow (Supplementary Fig. 59), highlighting the importance of strong phosphorescent units for the formation of afterglow luminophore-protein complex. Since free NAPpBP in aqueous system didn’t show afterglow at freezing temperature (as can be seen in the case of NAPpBP-PEG, Supplementary Fig. 55), we can simplify the experimental procedure by directly adding powders of NAPpBP-Biotin into aqueous solution of streptavidin. After stirring the mixture for 8h, the NAPpBP-Biotin powders disappear, which suggest the NAPpBP-Biotin molecules gradually dissolved in the protein solution driven by the strong SA-Biotin binding. After being stored at -20 °C, the frozen NAPpBP-Biotin-SA sample also exhibit significant afterglow (Supplementary Fig. 60). This protocol avoid the use of organic solvent and eliminate separation procedures. Based on these findings, we propose that phosphorescent molecules modified with specific moieties possess significant potential for the specific recognition of proteins. To the best of our knowledge on the function and application of organic afterglow materials, our study represents an interesting example of specific recognition and turn-on sensing of biomacromolecules by organic afterglow emitters. Despite the situation that the technology of such specific recognition and sensing is not mature, it still represents the initial step of organic afterglow materials towards corresponding biological application fields.

During revising this manuscript, we discuss with our corporate partners and perform more studies regarding the function or application of the current finding. One of our corporate partners, Shandong Longchang Animal Health Product Co., Ltd., which is a global leader in the bile acids industry, is expanding the application scenarios of its bile acid products in the biomedical field. A critical task is to identify target proteins whose activity can be controlled by bile acids. Here we build a model compound named NAPBP-CDCA by covalently linking chenodeoxycholic acid (CDCA) to NAPBP phosphorescent moiety (Fig. 10a); the synthetic details and structural characterization results of the NAPBP-CDCA can be found in Supplementary Information. After incubation of NAPBP-CDCA and lipase (porcine pancreas) in aqueous solution at room temperature for 8h, the resultant mixture at freezing temperature has been found to exhibit significant afterglow that originates from the long-lived phosphorescence of NAPBP moiety (Fig. 10b–d). In control experiment, neither the aqueous suspension of NAPBP compounds nor the mixture of NAPBP and lipase in aqueous medium show afterglow at freezing temperature (Fig. 10b). With reference to the results in NAPBP-Biotin-SA system, such observation in NAPBP-CDCA-lipase system suggest some non-covalent association, binding or recognition between CDCA and lipase; in this way, NAPBP moiety would deposit in some hydrophobic domain of lipase and its T1 state can be well protected by lipase at freezing temperature, which can explain the emergence of significant afterglow in this NAPBP-CDCA-lipase system. With this information, we test lipase activity in the presence of CDCA. It has been found that 594 μmol of fatty acid can be generated from olive oil per hour at 37 °C and at pH 8.3 (5% NaHCO3) by using 1 mg/mL lipase in the presence of 10 mg/mL CDCA (Fig. 10e). Such amount of fatty acid generated is much larger than that in the control experiment without the addition of CDCA (158 μmol). These results not only demonstrate that CDCA can significantly enhance lipase activity (which is very important for animal health and growth) but also indicate that our technology based on afterglow ice provides a visual pathway to identify target proteins for specific biologically active compounds. Further studies are still in progress, which would enlarge the application scope of bile acids in biomedical areas in the near future.

Methods
Preparation of organic RTP materials by doping with BA matrix
For the preparation of phosphor-BA-0.1% materials, 100 μL phosphorescent molecules in acetone (1 mg/mL) and 100 mg benzoic acid (BA) were first added to an agate mortar at a weight ratio of 1:1000 and were mixed by grinding with the aid of a mixed solvent of acetone-ethanol. The mixed solvent of acetone-ethanol, which can dissolve both phosphorescent molecules and BA, assists the mixing of phosphorescent molecules and BA. After grinding and solvent evaporation, the organic two-component afterglow materials was obtained.

Preparation of luminophore-protein conjugates
In an 8 mM sodium bicarbonate solution, the phosphorescent molecule was mixed with the protein. The resulting mixture was stirred at 25 °C for 2h to ensure adequate interaction between the phosphorescent molecule and the protein. Following the incubation period, the mixture was subjected to dialysis to remove any unbound phosphorescent molecules and other low molecular weight impurities. The dialysis was conducted over a period of 48h, during which the solution was dialyzed four times, each time against fresh deionized water. This process allowed for the gradual purification of the luminophore-protein conjugates, yielding a highly purified dialysate containing the desired complex. The resulting luminophore-protein conjugates was then collected.

Preparation of organic afterglow protein microspheres assisted by polyelectrolyte binder
The polyelectrolyte (either polyethyleneimine or poly-L-arginine) and carboxylated PS microspheres were stirred at 25 °C for 30min to facilitate the modification of the microsphere surface with the polyelectrolyte. Afterward, the reaction mixture was subjected to centrifugation to remove the supernatant, which contained any unbound polyelectrolytes. The polyelectrolyte-modified microspheres were then immediately resuspended in a solution containing 2COOHNAPBF2-BSA conjugates. The suspension was allowed to stand at room temperature for an additional 30min, facilitating the electrostatic attachment of the 2COOHNAPBF2-BSA conjugate to the microsphere surface. To ensure the removal of any unreacted conjugates, the reaction mixture was subjected to four to five rounds of centrifugation. The obtained organic afterglow protein microspheres were then dispersed in water by using Pluronic F127, a triblock copolymer, to achieve a stable colloidal suspension.

Preparation of organic afterglow protein microspheres via NHS ester chemistry
N-Hydroxysuccinimide (NHS), 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride (EDC) and carboxylated microspheres were stirred at 25 °C for 30min to facilitate the modification of the microsphere surface with NHS ester. After the activation step, the reaction mixture was subjected to centrifugation to remove the supernatant, which contained any unreacted reagents. The activated microspheres were then immediately resuspended in a solution containing 2COOHNAPBF2-insulin conjugates. The suspension was allowed to react at room temperature for an additional 30min, facilitating the covalent attachment of the 2COOHNAPBF2-insulin conjugate to the microsphere surface. To ensure the removal of any unreacted conjugates, the reaction mixture was subjected to four to five rounds of centrifugation. The obtained organic afterglow protein microspheres were then dispersed in water by using Pluronic F127, a triblock copolymer, to achieve a stable colloidal suspension.

Preparation of Luminophore-Protein Complexes
In an 8 mM sodium bicarbonate solution, the phosphorescent molecule was mixed with the protein. The resulting mixture was stirred at 25 °C for 2h to ensure adequate interaction between the phosphorescent molecule and the protein. Following the incubation period, the mixture was subjected to dialysis to remove any unbound phosphorescent molecules and other low molecular weight impurities. The dialysis was conducted over a period of 13h, during which the solution was dialyzed four times, each time against fresh deionized water. This process allowed for the gradual purification of the luminophore-protein complex, yielding a highly purified dialysate containing the desired complex. The resulting luminophore-protein complex was then collected.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This manuscript reports an interesting study on the phenomenon termed afterglow ice, representing organic phosphorescence from aqueous solutions of luminophore-protein conjugates and complexes at freezing temperatures. The authors utilized established labeling methods, including NHS ester chemistry and innovative β-diketone-protein conjugation, to demonstrate the substantial afterglow emission. This work broadens the functional applicability of organic phosphorescence to bioanalytical contexts, introducing specific recognition and sensing of biomacromolecules by organic afterglow emitters. Although preliminary in practical application, the manuscript provides rigorous experiments, detailed controls, and insightful mechanistic explorations. I thus suggest it for publication in Nature Communications following some revisions. My detailed comments and suggestions are listed below:\n(1) Benzophenone is a well-established ISC-promoting moiety, and its incorporation into a molecular system typically enhances the intersystem crossing quantum yield while reducing the phosphorescence lifetime. However, in this study, the introduction of benzophenone into naphthoic acid results in a prolonged phosphorescence lifetime. The authors are encouraged to provide a more comprehensive explanation for this phenomenon.\n(2) The authors should provide an assessment of the biotoxicity of the luminophore-protein conjugates or complexes in this study.\n(3) The authors have developed a protein labeling method based on β-diketone molecules. I would appreciate a more detailed explanation of the advantages of this approach relative to conventional NHS ester labeling methods. A comparative analysis supported by experimental data or literature references would significantly strengthen the manuscript by highlighting the unique contributions and potential benefits of this new labeling strategy.\n(4) While the authors acknowledge that this technology is still immature for immediate biomedical applications, a more explicit discussion outlining the steps necessary to bridge current findings to practical bioanalytical methods would enrich the manuscript.", "reviewer_2": "This manuscript presents a pioneering concept—to the best of my knowledge, the first demonstration of organic phosphorescence (“afterglow”) from aqueous solutions of luminophore–protein conjugates and complexes at sub-zero temperatures. The authors aim to explore organic afterglow emissions in biomacromolecular systems and introduce a novel β-diketone-based chemical labeling approach. The combination of hydrophobic interactions, covalent bonding, and low-temperature stabilization is proposed to explain the observed afterglow phenomena.\nThe core of the work focuses on synthesizing new luminophores that can be covalently attached to proteins. The authors then carry out a well-designed set of control experiments to demonstrate that the luminophore tags are indeed covalently bound. The successful observation of long-lived phosphorescence is impressive and holds clear potential for future applications—congratulations!\nHowever, at this stage, the findings remain somewhat heuristic. While the manuscript presents compelling visual and spectroscopic evidence (e.g., UV-Vis, phosphorescence spectra, and control experiments) supporting the formation of luminophore–protein conjugates, it lacks direct, quantitative, or structural evidence for covalent or specific binding between the luminophores and the proteins. Moreover, the role of temperature and the physical state (i.e., the frozen aqueous environment) in enabling or enhancing phosphorescence is not sufficiently explored. Temperature-dependent measurements of afterglow lifetimes would strengthen the argument that the matrix (ice) plays an essential role. Apparently, the phosphorescence is related to actual conjugate formation but what are the other necessary conditions induced by low temperature? Could it be precipitation or phase separation?\nLastly, the term “afterglow ice” may be misleading, as it implies that the ice itself is the source of luminescence. A more precise term could better reflect the actual origin of the observed phenomenon.", "reviewer_3": "In this paper, Li et al. report an organic phosphorescence phenomenon from a series of luminophore-protein conjugates which show afterglow emission at frozen temperature and they termed afterglow ice for abbreviation. In addition, they also showed that β-diketone moiety can be used to label protein via the formation of enamine chemistry. While the authors have synthesized a series of fluorescent dye to showcase the phenomenon, I do not believe that the work is suitable for Nature Communications. The reasons are following;\n1. The potential applications of the afterglow ice phenomenon seem very limited, either in chemical sensing, therapeutics or others. As most of the biochemistry and chemical sensing are conducted at 37 C or room temperature. The authors should showcase one example that can explore the afterglow ice observation.\n2. The authors should compare the delayed emission spectra of the luminophore-protein conjugates at room temperature and at the frozen temperature.\n3. The authors should perform a temperature titration experiment to understand the temperature effect and describe in detail the mechanism. For example, what is the cause of this afterglow effect, restricted bond rotation in macromolecules, hydrophobic effect or temperature.\n4. The quantitative analysis of the afterglow ice luminophore-protein conjugates should be compared with other afterglow materials, not just only the time-resolved analysis.\n5. Protein labeling using β-diketone moiety should be compared with the NHS ester group to compare the labeling efficiency. Furthermore, the SDS-PAGE gel shown in Figure 3d is not very convincing due to the strong fluorescence background.\n6. The interaction of NAPpBP-Biotin with streptavidin to generate afterglow signals have to undergo separation and performed at the frozen temperature. While I understand that this is just a proof-of-principle demonstration, the protocol has limited the potential application of the molecular interaction.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response: We thank the reviewer for the very positive comments on our manuscript. Since the detailed suggestions are listed below, we make corresponding response in the following.\n(1) Benzophenone is a well-established ISC-promoting moiety, and its incorporation into a molecular system typically enhances the intersystem crossing quantum yield while reducing the phosphorescence lifetime. However, in this study, the introduction of benzophenone into naphthoic acid results in a prolonged phosphorescence lifetime. The authors are encouraged to provide a more comprehensive explanation for this phenomenon.\nResponse 1: We thank the reviewer for the valuable comments and suggestion. Benzophenone (BP) is recognized as a significant phosphorescent emitter, characterized by an intersystem crossing efficiency that approaches unity. However, it exhibits a notably short phosphorescence lifetime, typically ranging from 0.1 to 1 ms, primarily due to the pronounced n-π* transition inherent to its triplet state. Recent research indicates that advanced charge transfer technology can maintain strong intersystem crossing in benzophenone-derived organic systems and simultaneously reduce phosphorescence rate constant (k ) to small values[23,70]. When nonradiative decay pathways are restricted using a rigid environment, it becomes possible to achieve highly efficient and persistent room-temperature phosphorescence. The benzophenone moiety is electron-deficient. By connecting it with electron-donating aromatic groups, an intramolecular charge transfer (ICT) system is established. The donor group contributes actively to the triplet excited state formation of the resulting system. If the donating group possesses a low-energy T state of localized excitation (LE) character, the T state of the resulting molecules would consist of remarkable portion from the donating group’s T state. As a result, the resulting molecules’ T states would inherit the donating group’s LE character and thus have small k . Naphthoic acid features a relatively low T energy level; upon conjugation with the BP group, the resultant NAPBP-COOH molecule capitalizes on the localized excitation characteristics of naphthoic acid's triplet state, thereby facilitating the formation of a long-lived T state within NAPBP-COOH (Supplementary Fig. 18). This molecular design effectively preserves the robust intersystem crossing capability of the benzophenone framework while attenuating the contribution of the n-π* transition to the T state (Supplementary Fig. 18). Consequently, this configuration endows the luminophore with both high intersystem crossing efficiency and a small phosphorescence rate constant. The above explanation on the long phosphorescence lifetime of NAPBP-COOH system has been added in the main text of this revised manuscript.\n(2) The authors should provide an assessment of the biotoxicity of the luminophore-protein conjugates or complexes in this study.\nResponse 2: We thank the reviewer for the valuable suggestion. Accordingly, we perform MTT cytotoxicity assays in this revised manuscript, where the experimental details have been added in this revised supplementary information. NAPBP-BSA conjugates, 2COOHNAP-diketone-BSA conjugates, NAPBP-Biotin-SA complex have been selected for the cytotoxicity measurement (Supplementary Fig. 21, 34 and 58). The samples at low concentrations (2.5 mg/mL to 7.5 mg/mL) have negligible or low cytotoxicity to NIH3T3 cells. The cytotoxicity increases with sample concentration but is still acceptable at higher concentrations (10 mg/mL). These results have been added in this revised manuscript.\n(3) The authors have developed a protein labeling method based on β-diketone molecules. I would appreciate a more detailed explanation of the advantages of this approach relative to conventional NHS ester labeling methods. A comparative analysis supported by experimental data or literature references would significantly strengthen the manuscript by highlighting the unique contributions and potential benefits of this new labeling strategy.\nResponse 3: We thank the reviewer for the valuable suggestion. It is known that conventional NHS esters are moisture-sensitive and are recommended to be stored under an inert atmosphere. For the β-diketone-based method, the luminophors (either the β-diketone or the BF bdk) are relatively stable and allow long-term storage under ambient conditions. Besides, our research group builds a molecular library that now contains over 500 BF bdk compounds, from which we can choose luminophors with diverse phosphorescence colors and other desired properties. Moreover, most of the luminophores have intramolecular charge transfer property, which can be readily excited by UVA and visible lights, show strong intersystem crossing efficiency and modest phosphorescence decay rate. Consequently, the resultant luminophore-protein conjugates would exhibit significant phosphorescence property at freezing temperature. These contents have been added in the main text of the revised manuscript.\n(4) While the authors acknowledge that this technology is still immature for immediate biomedical applications, a more explicit discussion outlining the steps necessary to bridge current findings to practical bioanalytical methods would enrich the manuscript.\nResponse 4: We thank the reviewer for the valuable suggestion. We understand that this comment refers to the function or application of the current finding. Accordingly, we perform more studies in this revised manuscript. Our corporate partner, Shandong Longchang Animal Health Product Co., Ltd., which is a global leader in the bile acids industry, is expanding the application scenarios of its bile acid products in the biomedical field. A critical task is to identify target proteins whose activity can be controlled by bile acids. Here we build a model compound named NAPBP-CDCA by covalently linking chenodeoxycholic acid (CDCA) to NAPBP phosphorescent moiety; the synthetic details and structural characterization results of the NAPBP-CDCA can be found in Supplementary Information. After incubation of NAPBP-CDCA and lipase (porcine pancreas) in aqueous solution at room temperature for 8 h, the resultant mixture at freezing temperature has been found to exhibit significant afterglow that originates from the long-lived phosphorescence of NAPBP moiety (Fig. 9b, c and d). In control experiment, neither the aqueous suspension of NAPBP compounds nor the mixture of NAPBP and lipase in aqueous medium show afterglow at freezing temperature (Fig. 9b). With reference to the results in NAPpBP-Biotin-SA system, such observation in NAPBP-CDCA-lipase system suggest some non-covalent association, binding or recognition between CDCA and lipase; in this way, NAPBP moiety would deposit in some hydrophobic domain of lipase and its T state can be well protected by lipase at freezing temperature, which can explain the emergence of significant afterglow in this NAPBP-CDCA-lipase system. With this information, we test lipase activity in the presence of CDCA. It has been found that 594 μmol of fatty acid can be generated from olive oil per hour at 37 °C and at pH 8.3 (5% NaHCO ) by using 1 mg/mL lipase in the presence of 10 mg/mL CDCA (Fig. 9e). Such amount of fatty acid generated is much larger than that in the control experiment without the addition of CDCA (158 μmol). These results not only demonstrate that CDCA can significantly enhance lipase activity (which is very important for animal health and growth) but also indicate that our technology based on afterglow ice provides a visual pathway to identify target proteins for specific biologically active compounds. Further studies are still in progress, which would enlarge the application scope of bile acids in biomedical areas in the near future. These contents have been added in the main text of this revised manuscript.\nResponse: We thank the reviewer for the very positive and enthusiastic comments. Since the detailed comments and suggestion are listed below, we make corresponding response in a point-to-point manner in the following.\nComment 1: However, at this stage, the findings remain somewhat heuristic. While the manuscript presents compelling visual and spectroscopic evidence (e.g., UV-Vis, phosphorescence spectra, and control experiments) supporting the formation of luminophore–protein conjugates, it lacks direct, quantitative, or structural evidence for covalent or specific binding between the luminophores and the proteins.\nResponse 1: We thank the reviewer for the valuable comments and suggestion. Accordingly, we perform matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for pristine BSA and 1NAP-BSA conjugates. The average molecular weight of 1NAP-BSA conjugates (66960 Da) exceeds that of BSA (66531) by 429 Da (Supplementary Fig. 1), corresponding to the addition of approximately two 1NAP group. These results agree well with the feeding molar ratio of 1NAP-NHS/BSA (2/1) and the estimated labeling efficiency (95.6%) (regarding the details of labeling efficiency, please find the response to Reviewer 3’s Comment 5). It is known that MALDI-TOF works at relatively high energy, where weak bonds and non-covalent binding would be disrupted, so the molecular increase by 429 Da can be unambiguously attributed to the covalent linkage of 1NAP group to BSA through NHS ester chemistry.\nRegarding the covalent linkage between β-diketone and BSA, we use various small-molecule amines as models to test the chemistry. We divided 2COOHNAPBF into five groups under alkaline conditions: one untreated group and the remaining groups treated with various amines, including glycine (Gly), L-lysine (Lys), L-arginine (L-Arg), and n-butylamine. High-performance liquid chromatography with mass spectrometry (HPLC-MS) was employed to monitor the formation of β-diketone and β-enaminone products. The HPLC-MS results indicated that free 2COOHNAPBF generates a β-diketone with a m/z of 287 in mildly alkaline conditions (Supplementary Fig. 28). Additionally, β-diketone peaks and corresponding β-enaminone peaks were observed for all four amine-treated systems (Supplementary Fig. 29 and S30), confirming that β-diketones can react with amine groups to produce β-enaminones. Moreover, we aimed to further establish the feasibility of β-diketone-based protein labeling through SDS-PAGE electrophoresis. Following reactions of five BF bdk compounds with BSA in alkaline conditions and subsequent dialysis, we obtained five BF bdk-BSA aqueous solutions exhibiting fluorescence when excited at 365 nm. These were tested alongside a control group of pristine BSA in SDS-PAGE. All six samples exhibited similar migration patterns under daylight. Under 365 nm excitation, the BF bdk-BSA solutions showed fluorescence, while the pristine BSA didn’t exhibit any fluorescence (Fig. 3d). This result corroborates the ability of β-diketones to achieve covalent labeling of proteins.\nThese contents have been highlighted in the main text of this revised manuscript.\nComment 2: Moreover, the role of temperature and the physical state (i.e., the frozen aqueous environment) in enabling or enhancing phosphorescence is not sufficiently explored. Temperature-dependent measurements of afterglow lifetimes would strengthen the argument that the matrix (ice) plays an essential role.\nResponse 2: We thank the reviewer for the valuable comments and suggestion. Accordingly, we collect temperature-dependent delayed emission spectra and emission decay profiles for NAPBP-BSA and 2COOHNAP-diketone-BSA conjugates (Supplementary Fig. 17 and 25). The delayed emission intensity is very weak at 278 K because the active nonradiative decay and oxygen quenching of triplet excited states in the fluidic aqueous solution. Upon freezing, the delayed emission intensity has significant increase, indicating the essential role of ice for the protection of luminophore’s triplet excited states. It has also been found that, by further decreasing the temperature, the delayed emission intensity and phosphorescence lifetimes increase, which can be attributed to better protection of luminophore’s triplet excited states within the ice at lower temperature. These contents have been added in this revised manuscript.\nComment 3: Apparently, the phosphorescence is related to actual conjugate formation but what are the other necessary conditions induced by low temperature? Could it be precipitation or phase separation?\nResponse 3: We thank the reviewer for the valuable comments. We understand that some compounds may precipitate at low temperature. In this revised manuscript, the aqueous solution of NAPBP-BSA conjugates are immediately frozen by liquid nitrogen and then placed in -20 °C refrigerator for one day. The sample shows homogenous afterglow upon removing excitation source (Supplementary Fig. 19). Besides, the NAPBP-BSA conjugates have been diluted to 1 mg/mL. The frozen solution of this diluted sample still shows significant afterglow (Supplementary Fig. 20). Since precipitation or phase separation would unlikely occur during rapid freezing and for samples at low concentration, these results can rule out afterglow mechanism by precipitation or phase separation, and thus support our proposed mechanism of synergistic effects of conjugate formation and hydrophobic interactions at freezing temperature for the emergence of afterglow. These results and discussion have been added in this revised manuscript.\nComment 4: Lastly, the term “afterglow ice” may be misleading, as it implies that the ice itself is the source of luminescence. A more precise term could better reflect the actual origin of the observed phenomenon.\nResponse 4: We thank the reviewer for the valuable comments. We respectfully acknowledge the concern regarding potential ambiguity in the term “afterglow ice.” However, we would like to clarify that this term is explicitly introduced and clearly defined in the abstract of our manuscript, where we state: “the first observation of organic afterglow from aqueous solutions of luminophore-protein conjugates and complexes at freezing temperature, named as afterglow ice for abbreviation.” This phrasing makes it clear that the luminescence originates from the luminophore-protein systems embedded within the ice matrix, rather than the ice itself. Throughout the manuscript, the term “afterglow ice” is consistently used to refer to this specific phenomenon. Given that the term is clearly defined at the beginning, we believe it does not introduce any significant risk of misinterpretation. Moreover, as a novel phenomenon, “afterglow ice” serves not only as a descriptive term but also as a concise label for the breakthrough itself—facilitating clear communication of our findings within the scientific community. These contents have been added as Supporting Text S1 in Supplementary Information.\nResponse: We thank the reviewer for the valuable comments. Since the detailed comments are listed below, we make corresponding response in the following.\nComment 1. The potential applications of the afterglow ice phenomenon seem very limited, either in chemical sensing, therapeutics or others. As most of the biochemistry and chemical sensing are conducted at 37 C or room temperature. The authors should showcase one example that can explore the afterglow ice observation.\nResponse 1: We thank the reviewer for the valuable comments and suggestion. During revising this manuscript, we discuss with our corporate partners and perform more studies regarding the function or application of the current finding of the afterglow ice phenomenon. One of our corporate partners, Shandong Longchang Animal Health Product Co., Ltd., which is a global leader in the bile acids industry, is expanding the application scenarios of its bile acid products in the biomedical field. A critical task is to identify target proteins whose activity can be controlled by bile acids. Here we build a model compound named NAPBP-CDCA by covalently linking chenodeoxycholic acid (CDCA) to NAPBP phosphorescent moiety (Fig. 9a); the synthetic details and structural characterization results of the NAPBP-CDCA can be found in Supplementary Information. After incubation of NAPBP-CDCA and lipase (porcine pancreas) in aqueous solution at room temperature for 8 h, the resultant mixture at freezing temperature has been found to exhibit significant afterglow that originates from the long-lived phosphorescence of NAPBP moiety (Fig. 9b, c and d). In control experiment, neither the aqueous suspension of NAPBP compounds nor the mixture of NAPBP and lipase in aqueous medium show afterglow at freezing temperature (Fig. 9b). With reference to the results in NAPBP-Biotin-SA system, such observation in NAPBP-CDCA-lipase system suggest some non-covalent association, binding or recognition between CDCA and lipase; in this way, NAPBP moiety would deposit in some hydrophobic domain of lipase and its T state can be well protected by lipase at freezing temperature, which can explain the emergence of significant afterglow in this NAPBP-CDCA-lipase system. With this information, we test lipase activity in the presence of CDCA. It has been found that 594 μmol of fatty acid can be generated from olive oil per hour at 37 °C and at pH 8.3 (5% NaHCO ) by using 1 mg/mL lipase in the presence of 10 mg/mL CDCA (Fig. 9e). Such amount of fatty acid generated is much larger than that in the control experiment without the addition of CDCA (158 μmol). These results not only demonstrate that CDCA can significantly enhance lipase activity (which is very important for animal health and growth) but also indicate that our technology based on afterglow ice provides a visual pathway to identify target proteins for specific biologically active compounds. Further studies are still in progress, which would enlarge the application scope of bile acids in biomedical areas in the near future. These contents have been added in the main text of this revised manuscript.\nComment 2. The authors should compare the delayed emission spectra of the luminophore-protein conjugates at room temperature and at the frozen temperature.\nResponse 2: We thank the reviewer for the valuable comments and suggestion. Accordingly, we collect the delayed emission spectra (1 ms delay) of NAPBP-BSA and 2COOHNAP-diketone-BSA conjugates at room temperature and frozen temperature (Supplementary Fig. 16 and 26). The aqueous solution of the conjugates at room temperature shows negligible delayed emission signals, which can be explained by the active nonradiative decay and oxygen quenching of luminophore’s triplet excited states at fluidic aqueous solution. At the frozen temperature, the nonradiative decay of luminophore’s triplet excited states can be suppressed within ice matrix and the oxygen diffusion can be inhibited by the ice matrix, leading to the significant phosphorescence (Supplementary Fig. 16 and 26).\nComment 3. The authors should perform a temperature titration experiment to understand the temperature effect and describe in detail the mechanism. For example, what is the cause of this afterglow effect, restricted bond rotation in macromolecules, hydrophobic effect or temperature.\nResponse 3: We thank the reviewer for the valuable comments and suggestion. Accordingly, we collect temperature-dependent delayed emission spectra and emission decay profiles for NAPBP-BSA and 2COOHNAP-diketone-BSA conjugates (Supplementary Fig. 17 and 25). The delayed emission intensity is very weak at 278 K because the active nonradiative decay and oxygen quenching of triplet excited states in the fluidic aqueous solution. Upon freezing, the delayed emission intensity has significant increase, indicating the essential role of ice for the protection of luminophore’s triplet excited states. It has also been found that, by further decreasing the temperature, the delayed emission intensity and phosphorescence lifetimes increase, which can be attributed to better protection of luminophore’s triplet excited states within the ice at lower temperature.\nBesides temperature, we also investigate the contribution of hydrophobic effect on the afterglow property. Sodium dodecyl sulfonate (SDS) is known to disrupt hydrophobic interactions in protein systems. In this revised manuscript, we add SDS into the aqueous solution of 2COOHNAP-diketone-BSA conjugates. After being stored at -20 °C, the frozen sample with the presence of SDS shows much weaker phosphorescence than the frozen sample without SDS (Supplementary Fig. 27). This observation supports our proposed mechanism that hydrophobic interaction and covalent linkage between luminophore and protein can protect organic triplet excited states from quenching.\nIn addition, NMR technique is used to study the mobility of luminophore in different microenvironments. When 1NAP groups are covalently linked to PEG or ε-polylysine (EPL) chains, the resultant 1NAP-PEG and 1NAP-EPL samples in D O show sharp and well-resolved 1H NMR signals in the aromatic region (Supplementary Fig. 8 and 9). The 1NAP groups have free motion in D O to give sharp peaks, because the hydrophilic polymers (PEG and EPL) endow 1NAP groups with excellent solubility in D O. In contrast, 1H NMR spectrum of 1NAP-BSA conjugates in D O shows the disappearance of NMR signals of 1NAP groups in the aromatic region (Supplementary Fig. 10), which suggest the motion of 1NAP groups is seriously restricted within BSA (Macromolecules 2003, 36, 2576-2578). The hydrophobic 1NAP groups within some hydrophobic microregion of BSA have largely restricted motion, which can significant reduce nonradiative decay of 1NAP’s triplet excited states, leading to the emergence of afterglow at freezing temperature.\nThe above results and discussion that further support our proposed afterglow mechanism have been added in this revised manuscript.\nComment 4. The quantitative analysis of the afterglow ice luminophore-protein conjugates should be compared with other afterglow materials, not just only the time-resolved analysis.\nResponse 4: We thank the reviewer for the valuable comments and suggestion. We understand that this reviewer encourages us to enhance the performance of afterglow ice and make a comparison with other related afterglow materials. In this revised manuscript, we apply deuteration technique to increase phosphorescence lifetimes of luminophore-protein conjugates. C-D stretching vibration is much weaker than C-H stretching vibration. Deuteration of luminophores can reduce the energy of effective nonclassical vibrational mode involved in the nonradiative transition, decrease Franck-Condon factor, and consequently reduce nonradiative decay, leading to the elongation of phosphorescence lifetime (Adv. Funct. Mater. 2013, 23, 3386–3397). Deuterated CorBF (DCorBF ) has been prepared and used to label BSA; the synthetic details can be found in Supplementary Information. The resultant aqueous solution of DCorBF -BSA conjugates at frozen state display phosphorescence lifetime of 2.29 s, which remains very rare in the related afterglow material systems (Fig. 5g and h). Supplementary Fig. 39 summarizes the reported afterglow materials in aqueous systems including supramolecular assemblies, suspension, emulsion and hydrogels, from which one can find that it is challenging to prepare materials with long phosphorescence lifetimes because of the active nonradiative decay and oxygen quenching in aqueous systems. Compared to these reported studies, the phosphorescence lifetimes of our DCorBF -BSA conjugates at frozen state are among the longest in the reported studies of aqueous afterglow systems. These contents have been added in this revised manuscript.\nComment 5. Protein labeling using β-diketone moiety should be compared with the NHS ester group to compare the labeling efficiency. Furthermore, the SDS-PAGE gel shown in Figure 3d is not very convincing due to the strong fluorescence background.\nResponse 5: We thank the reviewer for the valuable comments and suggestion. Accordingly, we estimate the labeling efficiency by UV-vis technique. For the NHS ester chemistry, the labeling efficiency is higher than 95% (Supplementary Fig. 2). In the case of β-diketone-based system, the labeling efficiency is 23.1% to 89.5% (Supplementary Fig. 35). It is known that conventional NHS esters are moisture-sensitive and are recommended to be stored under an inert atmosphere. For the β-diketone-based method, the luminophors (either the β-diketone or the BF bdk) are relatively stable and allow long-term storage under ambient conditions. Besides, our research group builds a molecular library that now contains over 500 BF bdk compounds, from which we can choose luminophors with diverse phosphorescence colors and other desired properties. Moreover, most of the luminophores have intramolecular charge transfer property, which can be readily excited by UVA and visible lights, show strong intersystem crossing efficiency and modest phosphorescence decay rate. Consequently, the resultant luminophore-protein conjugates would exhibit significant phosphorescence property at freezing temperature. These contents have been added in the main text of the revised manuscript.\nRegarding the SDS-PAGE gel shown in Figure 3d, we repeat the experiments for several times during revising this manuscript. The results have been added in the revised supplementary information, from which we can see the fluorescence from BF bdk-BSA conjugates (Supplementary Fig. 31). In order to remove the background of the SDS-PAGE gel, we use low-power UV excitation source, so that the fluorescence signals of BF bdk-BSA conjugates are weak in Supplementary Fig. 31. Therefore, in the main text, we still use the original Figure 3d that exhibit significant fluorescence of BF bdk-BSA conjugates. We hope the reviewer can understand that we have tried for many times to capture the photographs of SDS-PAGE gel under UV lamp. The photographs in the main text and supplementary information are the best results at the current stage.\nComment 6. The interaction of NAPpBP-Biotin with streptavidin to generate afterglow signals have to undergo separation and performed at the frozen temperature. While I understand that this is just a proof-of-principle demonstration, the protocol has limited the potential application of the molecular interaction.\nResponse 6: We thank the reviewer for the valuable comments and suggestion. Since free NAPpBP in aqueous system didn’t show afterglow at freezing temperature (as can be seen in the case of NAPpBP-PEG, Supplementary Fig. 55), we can simplify the experimental procedure by directly adding powders of NAPpBP-Biotin into aqueous solution of streptavidin. After stirring the mixture for 8 h, the NAPpBP-Biotin powders disappear, which suggest the NAPpBP-Biotin molecules gradually dissolved in the protein solution driven by the strong SA-Biotin binding. After being stored at -20 °C, the frozen NAPpBP-Biotin-SA sample also exhibit significant afterglow (Supplementary Fig. 60). This protocol avoids the use of organic solvent and eliminate separation procedures. These contents have been added in this revised manuscript. Regarding the potential application of the afterglow ice, please also refer to the response to Reviewer 3’s Comment 1.\nResponse: We thank the reviewer for the valuable comments and suggestion."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have carefully addressed all the concerns of the reviewers. This version is acceptable for publication.", "reviewer_3": "The authors have adequately addressed the reviewers' concerns. I recommend that the article be accepted for publication.", "reviewer_4": "Overall assessment\nThe authors have responded carefully to the majority of our comments. They provided new data (MALDI-TOF, HPLC-MS, SDS-PAGE) that strengthen the evidence for covalent labeling and partially added temperature-dependent emission studies clarifying the role of the frozen environment.\nRemaining concern – precipitation/phase separation\nHowever, one important issue remains insufficiently addressed. It is well established that only very small solutes can be incorporated into ice crystals, while proteins and protein–dye conjugates are excluded into brine channels during freezing, which often leads to precipitation or aggregation. The rebuttal relies on rapid freezing and dilution experiments to argue against precipitation, but these do not convincingly exclude micro- or nano-scale precipitation/phase separation. Visual “homogeneity” of luminescence is not sufficient evidence. Without direct demonstration, the mechanistic conclusion that the afterglow arises solely from “hydrophobic interaction and covalent linkage protected within ice” remains incomplete.\nRecommendation\nThe manuscript is otherwise strong and makes an important contribution. If the authors can provide more direct evidence to rule out precipitation or at least acknowledge it as a likely contributing factor, I would be fully supportive of publication.\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Response: We thank the reviewer for the positive comments and recommendation on our manuscript.\nResponse: We thank the reviewer for the positive comments and recommendation on our manuscript.\nResponse: We thank the reviewer for the valuable comments and positive recommendation on our manuscript. To further study whether protein precipitation/phase separation during freezing contributes to the emergence of afterglow, we covalently link PEG chains to NAPBP-BSA conjugates; the steric repulsion between PEG chains would minimize the possibility of protein aggregation especially in the case when the NAPBP-BSA conjugates are heavily decorated with PEG chains. After being frozen, the PEG-decorated NAPBP-BSA conjugates have been found to show similar afterglow properties compared to NAPBP-BSA conjugates (control sample, without PEG) (Supplementary Fig. 20D-Q). These studies show that protein aggregation may occur during freezing in the case of luminophore-protein conjugates but has insignificant contribution to afterglow emergence in the present system. These contents, which support our proposed mechanism of synergistic effects of conjugate formation and hydrophobic interactions at freezing temperature for the emergence of afterglow, have been added in this revised manuscript."}, {"round": 3, "reviewer_comments": {"reviewer_4": "Overall assessment\nThe authors have responded carefully to the majority of our comments. They provided new data (MALDI-TOF, HPLC-MS, SDS-PAGE) that strengthen the evidence for covalent labeling and partially added temperature-dependent emission studies clarifying the role of the frozen environment.\nRemaining concern – precipitation/phase separation\nHowever, one important issue remains insufficiently addressed. It is well established that only very small solutes can be incorporated into ice crystals, while proteins and protein–dye conjugates are excluded into brine channels during freezing, which often leads to precipitation or aggregation. The rebuttal relies on rapid freezing and dilution experiments to argue against precipitation, but these do not convincingly exclude micro- or nano-scale precipitation/phase separation. Visual “homogeneity” of luminescence is not sufficient evidence. Without direct demonstration, the mechanistic conclusion that the afterglow arises solely from “hydrophobic interaction and covalent linkage protected within ice” remains incomplete.\nRecommendation\nThe manuscript is otherwise strong and makes an important contribution. If the authors can provide more direct evidence to rule out precipitation or at least acknowledge it as a likely contributing factor, I would be fully supportive of publication.\nThe authors have adequately addressed the reviewers' concerns. I think that the article can be accepted for publication."}, "author_response": ""}]

Decision letter