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Molecular rotor-based probes for protein monitoring in biomedical research

Nature Reviews Chemistry volume 10, pages 350–366 (2026) Cite this article

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Abstract

As the fundamental executors of biological function, proteins are frequently dysregulated or differentially expressed in disease states, making them valuable biomarkers and/or therapeutic targets. Conventional approaches to monitoring the presence and activity of these proteins — including enzyme-linked immunosorbent assay, western blotting and mass spectrometry — have limited ability to provide real-time information on living cells. Fluorescence imaging overcomes these limitations by enabling selective, non-invasive and dynamic protein analysis. Molecular rotor fluorophores offer unique advantages owing to their high sensitivity to microenvironmental changes and tunable photophysical properties. Notably, these molecular rotor scaffolds can be functionalized into targeting probes that become highly emissive upon binding to specific proteins via the restriction of intramolecular rotation. Here, we introduce molecular rotor-based probes and outline their design principles and detection mechanisms. We highlight their applications in disease diagnosis and biological research, and we discuss the current challenges and prospects for clinical translation.

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Key points

  • Molecular rotor-based probes enable real-time and non-invasive protein monitoring in living cells and organisms by fluorescing upon binding, overcoming limitations of traditional fixed-sample methods such as enzyme-linked immunosorbent assay and western blotting.

  • The unique ‘light-up’ mechanism relies on restriction of intramolecular rotation when the rotor binds to target proteins, providing high signal-to-noise and microenvironment sensitivity.

  • Molecular rotor scaffolds can be functionalized via covalent tagging, ligand-directed targeting or enzyme-activatable strategies to achieve high specificity for proteins, aggregates or proteolytic activity.

  • These probes support diverse biomedical applications and fundamental research, including tracking misfolded proteins in neurodegenerative diseases, visualizing oncogenic signatures for cancer diagnosis, guiding fluorescence-assisted surgery, elucidating protein–protein interactions, conformational dynamics and localization in complex native environments.

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Fig. 1: Design strategies and signal regulation mechanisms of molecular rotor-based probes for protein monitoring.
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Fig. 2: Diagnosis of neurodegenerative disease.
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Fig. 3: Molecular rotor-based probe for protein monitoring in cancer diagnosis.
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Fig. 4: Guided surgery and therapy.
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Fig. 5: Molecular rotor-based probes for analysis of protein–protein interactions.
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Fig. 6: Protein–DNA/RNA interactions.
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Fig. 7: Application of molecular rotor-based protein in target identification for proteomics.
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References

  1. Tang, J. et al. Longitudinal serum proteome mapping reveals biomarkers for healthy ageing and related cardiometabolic diseases. Nat. Metab. 7, 166–181 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Ali, M. et al. Shared and disease-specific pathways in frontotemporal dementia and Alzheimer’s and Parkinson’s diseases. Nat. Med. 31, 2567–2577 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Liu, J., Cheng, P., Xu, C. & Pu, K. Molecular probes for in vivo optical imaging of immune cells. Nat. Biomed. Eng. 9, 618–637 (2025).

    Article  PubMed  Google Scholar 

  4. Minoshima, M., Reja, S. I., Hashimoto, R., Iijima, K. & Kikuchi, K. Hybrid small-molecule/protein fluorescent probes. Chem. Rev. 124, 6198–6270 (2024).

    Article  CAS  PubMed  Google Scholar 

  5. Wu, X., Wang, R., Kwon, N., Ma, H. & Yoon, J. Activatable fluorescent probes for in situ imaging of enzymes. Chem. Soc. Rev. 51, 450–463 (2022).

    Article  CAS  PubMed  Google Scholar 

  6. Kubota, R. & Hamachi, I. Protein recognition using synthetic small-molecular binders toward optical protein sensing in vitro and in live cells. Chem. Soc. Rev. 44, 4454–4471 (2015).

    Article  CAS  PubMed  Google Scholar 

  7. Cohen, L. & Walt, D. R. Highly sensitive and multiplexed protein measurements. Chem. Rev. 119, 293–321 (2018).

    Article  PubMed  Google Scholar 

  8. Zhao, L. & Miao, Q. Organic afterglow luminescence for disease diagnosis and treatment. Nat. Rev. Bioeng. 3, 955–975 (2025).

    Article  CAS  Google Scholar 

  9. Yan, D., Wang, D. & Tang, B. Z. In vivo, clinical and translational aspects of aggregation-induced emission. Nat. Rev. Bioeng. 3, 976–991 (2025).

    Article  Google Scholar 

  10. Ye, S., Hsiung, C.-H., Tang, Y. & Zhang, X. Visualizing the multistep process of protein aggregation in live cells. Acc. Chem. Res. 55, 381–390 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Wang, M., Da, Y. & Tian, Y. Fluorescent proteins and genetically encoded biosensors. Chem. Soc. Rev. 52, 1189–1214 (2023).

    Article  CAS  PubMed  Google Scholar 

  12. Van Roessel, P. & Brand, A. H. Imaging into the future: visualizing gene expression and protein interactions with fluorescent proteins. Nat. Cell Biol. 4, E15–E20 (2002).

    Article  PubMed  Google Scholar 

  13. Arsić, A., Hagemann, C., Stajković, N., Schubert, T. & Nikić-Spiegel, I. Minimal genetically encoded tags for fluorescent protein labeling in living neurons. Nat. Commun. 13, 314 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Jain, R. K. Physiological barriers to delivery of monoclonal antibodies and other macromolecules in tumors. Cancer Res. 50, 814s–819s (1990).

    CAS  PubMed  Google Scholar 

  15. Zhao, C. et al. Vesicular antibodies: shedding light on antibody therapeutics with cell membrane nanotechnology. Adv. Mater. 35, 2207875 (2023).

    Article  CAS  Google Scholar 

  16. Mizukami, S., Hori, Y. & Kikuchi, K. Small-molecule-based protein-labeling technology in live cell studies: probe-design concepts and applications. Acc. Chem. Res. 47, 247–256 (2014).

    Article  CAS  PubMed  Google Scholar 

  17. Liu, H.-W. et al. Recent progresses in small-molecule enzymatic fluorescent probes for cancer imaging. Chem. Soc. Rev. 47, 7140–7180 (2018).

    Article  CAS  PubMed  Google Scholar 

  18. Grimm, J. B. & Lavis, L. D. Caveat fluorophore: an insiders’ guide to small-molecule fluorescent labels. Nat. Methods 19, 149–158 (2022).

    Article  CAS  PubMed  Google Scholar 

  19. Chan, J., Dodani, S. C. & Chang, C. J. Reaction-based small-molecule fluorescent probes for chemoselective bioimaging. Nat. Chem. 4, 973–984 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Zhang, J., Campbell, R. E., Ting, A. Y. & Tsien, R. Y. Creating new fluorescent probes for cell biology. Nat. Rev. Mol. Cell Biol. 3, 906–918 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Hu, L. et al. Designing artificial fluorescent proteins and biosensors by genetically encoding molecular rotor-based amino acids. Nat. Chem. 16, 1960–1971 (2024).

    Article  CAS  PubMed  Google Scholar 

  22. Yang, S. et al. Real-time imaging of protein microenvironment changes in cells with rotor-based fluorescent amino acids. Nat. Chem. Biol. 22, 97–108 (2026).

    Article  CAS  PubMed  Google Scholar 

  23. Xie, D. et al. A fluorescent molecular rotor for biomolecular imaging analysis. Chem. Commun. 61, 10408–10417 (2025).

    Article  CAS  Google Scholar 

  24. Bai, Y. et al. Advanced techniques for detecting protein misfolding and aggregation in cellular environments. Chem. Rev. 123, 12254–12311 (2023).

    Article  CAS  PubMed  Google Scholar 

  25. Ma, J. et al. Design and application of fluorescent probes to detect cellular physical microenvironments. Chem. Rev. 124, 1738–1861 (2024).

    Article  CAS  PubMed  Google Scholar 

  26. Wang, K. et al. Fluorescence image-guided tumour surgery. Nat. Rev. Bioeng. 1, 161–179 (2023).

    Article  CAS  Google Scholar 

  27. Wu, X., Li, H., Lee, E. & Yoon, J. Sensors for in situ real-time fluorescence imaging of enzymes. Chem 6, 2893–2901 (2020).

    Article  CAS  Google Scholar 

  28. Chung, H. K. & Lin, M. Z. On the cutting edge: protease-based methods for sensing and controlling cell biology. Nat. Methods 17, 885–896 (2020).

    Article  CAS  PubMed  Google Scholar 

  29. Wu, L., Huang, J., Pu, K. & James, T. D. Dual-locked spectroscopic probes for sensing and therapy. Nat. Rev. Chem. 5, 406–421 (2021).

    Article  CAS  PubMed  Google Scholar 

  30. Xu, C. & Pu, K. Artificial urinary biomarker probes for diagnosis. Nat. Rev. Bioeng. 2, 425–441 (2024).

    Article  CAS  Google Scholar 

  31. Cheng, P. & Pu, K. Molecular imaging and disease theranostics with renal-clearable optical agents. Nat. Rev. Mater. 6, 1095–1113 (2021).

    Article  CAS  Google Scholar 

  32. Paez-Perez, M. & Kuimova, M. K. Molecular rotors: fluorescent sensors for microviscosity and conformation of biomolecules. Angew. Chem. Int. Ed. 136, e202311233 (2024).

    Article  Google Scholar 

  33. Klymchenko, A. S. Solvatochromic and fluorogenic dyes as environment-sensitive probes: design and biological applications. Acc. Chem. Res. 50, 366–375 (2017).

    Article  CAS  PubMed  Google Scholar 

  34. Yin, J. et al. Small molecule based fluorescent chemosensors for imaging the microenvironment within specific cellular regions. Chem. Soc. Rev. 50, 12098–12150 (2021).

    Article  CAS  PubMed  Google Scholar 

  35. Wang, S. et al. Fluorescence imaging of pathophysiological microenvironments. Chem. Soc. Rev. 50, 8887–8902 (2021).

    Article  CAS  PubMed  Google Scholar 

  36. Haidekker, M. A. & Theodorakis, E. A. Environment-sensitive behavior of fluorescent molecular rotors. J. Biol. Eng. 4, 11 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Kuimova, M. K. Mapping viscosity in cells using molecular rotors. Phys. Chem. Chem. Phys. 14, 12671–12686 (2012).

    Article  CAS  PubMed  Google Scholar 

  38. Wang, C. et al. Twisted intramolecular charge transfer (TICT) and twists beyond TICT: from mechanisms to rational designs of bright and sensitive fluorophores. Chem. Soc. Rev. 50, 12656–12678 (2021).

    Article  CAS  PubMed  Google Scholar 

  39. Michel, B. Y., Dziuba, D., Benhida, R., Demchenko, A. P. & Burger, A. Probing of nucleic acid structures, dynamics, and interactions with environment-sensitive fluorescent labels. Front. Chem. 8, 112 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Chen, G. et al. Reactivity of functional groups on the protein surface: development of epoxide probes for protein labeling. J. Am. Chem. Soc. 125, 8130–8133 (2003).

    Article  CAS  PubMed  Google Scholar 

  41. Miao, W., Yu, C., Hao, E. & Jiao, L. Functionalized BODIPYs as fluorescent molecular rotors for viscosity detection. Front. Chem. 7, 825 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Naghibi, S., Chen, T., Jamshidi Ghahfarokhi, A. & Tang, Y. AIEgen-enhanced protein imaging: probe design and sensing mechanisms. Aggregate 2, e41 (2021).

    Article  CAS  Google Scholar 

  43. Xia, F. et al. Modular design of peptide- or DNA-modified AIEgen probes for biosensing applications. Acc. Chem. Res. 52, 3064–3074 (2019).

    Article  CAS  PubMed  Google Scholar 

  44. Wu, F. et al. Biomacromolecule-functionalized AIEgens for advanced biomedical studies. Small 15, e1804839 (2019).

    Article  PubMed  Google Scholar 

  45. Wu, X. et al. Recent progresses of peptide fluorescent probes for protein analysis in living cells. ACS Mater. Lett. 7, 2524–2533 (2025).

    Article  CAS  Google Scholar 

  46. Kalarikkal, C., Bhattacharjee, S. & Mapa, K. Lipid droplet specific BODIPY based rotors with viscosity sensitivity to distinguish normal and cancer cells: impact of molecular conformation. J. Mater. Chem. B 13, 1474–1486 (2025).

    Article  CAS  PubMed  Google Scholar 

  47. Venkatesh, Y., Marotta, N. P., Lee, V. M.-Y. & Petersson, E. J. Highly tunable bimane-based fluorescent probes: design, synthesis, and application as a selective amyloid binding dye. Chem. Sci. 15, 6053–6063 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kumar, B., Ghosh, R., Mora, A. K. & Nath, S. Anthryl benzothiazolium molecular rotor-based turn-on DNA probe: detailed mechanistic studies. J. Phys. Chem. B 123, 7518–7527 (2019).

    Article  CAS  PubMed  Google Scholar 

  49. Mu, X. et al. A cyanine-derived near-infrared molecular rotor for ratiometric imaging of mitochondrial viscosity in cells. Sens. Actuators B Chem. 298, 126831 (2019).

    Article  CAS  Google Scholar 

  50. Mu, X. et al. A cyanine-derived NIR molecular rotor for ratiometric imaging of amyloid-β aggregates. Sens. Actuators B Chem. 338, 129842 (2021).

    Article  CAS  Google Scholar 

  51. Wang, Y.-N. et al. Viscosity sensitive fluorescent dyes with excellent photostability based on hemicyanine dyes for targeting cell membrane. Sens. Actuators B Chem. 337, 129787 (2021).

    Article  CAS  Google Scholar 

  52. Zhang, X. et al. Design and screening of fluorescent probes based upon hemicyanine dyes for monitoring mitochondrial viscosity in living cells. J. Phys. Chem. B 128, 3910–3918 (2024).

    Article  CAS  PubMed  Google Scholar 

  53. Mukherjee, T. et al. Near infrared emitting molecular rotor based on merocyanine for probing the viscosity of cellular lipid environments. Mater. Chem. Front. 5, 2459–2469 (2021).

    Article  CAS  Google Scholar 

  54. Feng, D. et al. pH-/viscosity-activatable NIR fluorescent probes via acceptor engineering of hemicyanine dyes for high-contrast bioimaging. Anal. Chem. 97, 4041–4048 (2025).

    Article  CAS  PubMed  Google Scholar 

  55. Paige, J. S., Wu, K. Y. & Jaffrey, S. R. RNA mimics of green fluorescent protein. Science 333, 642–646 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Ye, S., Zhang, H., Fei, J., Wolstenholme, C. H. & Zhang, X. A general strategy to control viscosity sensitivity of molecular rotor-based fluorophores. Angew. Chem. Int. Ed. 60, 1339–1346 (2021).

    Article  CAS  Google Scholar 

  57. Sun, R. et al. Derivatizing Nile Red fluorophores to quantify the heterogeneous polarity upon protein aggregation in the cell. Chem. Commun. 58, 5407–5410 (2022).

    Article  CAS  Google Scholar 

  58. Ademoye, T. A. et al. In vitro evaluation of amide-linked coumarin scaffolds for the inhibition of α-synuclein and tau aggregation. ACS Omega 10, 38498–38514 (2025).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Carroll, E. C. et al. High-throughput discovery of fluoroprobes that recognize amyloid fibril polymorphs. Nat. Chem. 17, 1565–1575 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Huang, X. et al. Precise photorelease in living cells by high-viscosity activatable coumarin-based photocages. Chem. Sci. 16, 3611–3619 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Liu, Y. et al. A small molecule antagonist of SMN disrupts the interaction between SMN and RNAP II. Nat. Commun. 13, 5453 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Feng, L. et al. A fluorescent molecular rotor probe for tracking plasma membranes and exosomes in living cells. Chem. Commun. 56, 8480–8483 (2020).

    Article  CAS  Google Scholar 

  63. Zhang, J. et al. A prostate-specific membrane antigen activated molecular rotor for real-time fluorescence imaging. Nat. Commun. 12, 5460 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Hsu, Y.-P. et al. Fluorogenic D-amino acids enable real-time monitoring of peptidoglycan biosynthesis and high-throughput transpeptidation assays. Nat. Chem. 11, 335–341 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Dong, J. et al. Ultrathin two-dimensional porous organic nanosheets with molecular rotors for chemical sensing. Nat. Commun. 8, 1142 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  66. Sabouri, S. et al. Construction of a highly sensitive thiol-reactive AIEgen-peptide conjugate for monitoring protein unfolding and aggregation in cells. Adv. Healthc. Mater. 10, 2101300 (2021).

    Article  CAS  Google Scholar 

  67. Xu, L. et al. Naphthalene anhydride triphenylamine as a viscosity-sensitive molecular rotor for liquid safety inspection. N. J. Chem. 46, 3078–3082 (2022).

    Article  CAS  Google Scholar 

  68. Zhao, Z., He, B. & Tang, B. Z. Aggregation-induced emission of siloles. Chem. Sci. 6, 5347–5365 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Scalise, R. E., Caradonna, P. A., Tracy, H. J., Mullin, J. L. & Keirstead, A. E. 1,1-Dimethyl-2,3,4,5-tetraphenylsilole as a molecular rotor probe to investigate the microviscosity of imidazolium ionic liquids. J. Inorg. Organomet. Polym. Mater. 24, 431–441 (2014).

    Article  CAS  Google Scholar 

  70. Tang, H. et al. Arylamino-substituted rhodamine as a fluorogenic molecular rotor for the wash-free imaging of non-catalytic proteins in live cells. Anal. Sens. 4, e202300037 (2024).

    CAS  Google Scholar 

  71. Su, D., Teoh, C. L., Wang, L., Liu, X. & Chang, Y.-T. Motion-induced change in emission (MICE) for developing fluorescent probes. Chem. Soc. Rev. 46, 4833–4844 (2017).

    Article  CAS  PubMed  Google Scholar 

  72. Katori, A., Azuma, E., Ishimura, H., Kuramochi, K. & Tsubaki, K. Fluorescent dyes with directly connected xanthone and xanthene units. J. Org. Chem. 80, 4603–4610 (2015).

    Article  CAS  PubMed  Google Scholar 

  73. Alamudi, S. H. & Chang, Y.-T. Advances in the design of cell-permeable fluorescent probes for applications in live cell imaging. Chem. Commun. 54, 13641–13653 (2018).

    Article  CAS  Google Scholar 

  74. Kumagai, T., Kinoshita, B., Hirashima, S., Sugiyama, H. & Park, S. Thiophene-extended fluorescent nucleosides as molecular rotor-type fluorogenic sensors for biomolecular interactions. ACS Sens. 8, 923–932 (2023).

    Article  CAS  PubMed  Google Scholar 

  75. Zhang, X., Huo, F., Zhang, Y., Yue, Y. & Yin, C. Dual-channel detection of viscosity and pH with a near-infrared fluorescent probe for cancer visualization. Analyst 147, 2470–2476 (2022).

    Article  CAS  PubMed  Google Scholar 

  76. Qian, H. et al. Suppression of Kasha’s rule as a mechanism for fluorescent molecular rotors and aggregation-induced emission. Nat. Chem. 9, 83–87 (2017).

    Article  CAS  PubMed  Google Scholar 

  77. Yan, C. et al. Fluorescence umpolung enables light-up sensing of N-acetyltransferases and nerve agents. Nat. Commun. 12, 3869 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Ohno, H., Sumitani, S., Sasaki, E., Yamada, S. & Hanaoka, K. Recent advances in fluorogenic probes based on twisted intramolecular charge transfer (TICT) for live-cell imaging. Chem. Commun. 61, 12871–12884 (2025).

    Article  CAS  Google Scholar 

  79. Wang, C. et al. Quantitative design of bright fluorophores and AIEgens by the accurate prediction of twisted intramolecular charge transfer (TICT). Angew. Chem. Int. Ed. 132, 10246–10258 (2020).

    Article  Google Scholar 

  80. Wang, C. et al. Monitoring amyloid aggregation via a twisted intramolecular charge transfer (TICT)-based fluorescent sensor array. Chem. Sci. 14, 4786–4795 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Hartl, F. U., Bracher, A. & Hayer-Hartl, M. Molecular chaperones in protein folding and proteostasis. Nature 475, 324–332 (2011).

    Article  CAS  PubMed  Google Scholar 

  82. Qiang, W., Yau, W.-M., Lu, J.-X., Collinge, J. & Tycko, R. Structural variation in amyloid-β fibrils from Alzheimer’s disease clinical subtypes. Nature 541, 217–221 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  83. Shahnawaz, M. et al. Discriminating α-synuclein strains in Parkinson’s disease and multiple system atrophy. Nature 578, 273–277 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Ross, C. A. & Poirier, M. A. Protein aggregation and neurodegenerative disease. Nat. Med. 10, S10–S17 (2004).

    Article  PubMed  Google Scholar 

  85. Wang, W., Nema, S. & Teagarden, D. Protein aggregation — pathways and influencing factors. Int. J. Pharm. 390, 89–99 (2010).

    Article  CAS  PubMed  Google Scholar 

  86. Salvadores, N., Shahnawaz, M., Scarpini, E., Tagliavini, F. & Soto, C. Detection of misfolded Aβ oligomers for sensitive biochemical diagnosis of Alzheimer’s disease. Cell Rep. 7, 261–268 (2014).

    Article  CAS  PubMed  Google Scholar 

  87. Shin, J. et al. Harnessing intramolecular rotation to enhance two-photon imaging of Aβ plaques through minimizing background fluorescence. Angew. Chem. Int. Ed. 131, 5704–5708 (2019).

    Article  Google Scholar 

  88. Verwilst, P. et al. Rational design of in vivo tau tangle-selective near-infrared fluorophores: expanding the BODIPY universe. J. Am. Chem. Soc. 139, 13393–13403 (2017).

    Article  CAS  PubMed  Google Scholar 

  89. Venkatesh, Y., Narayan, K. B., Baumgart, T. & Petersson, E. J. Strategic modulation of polarity and viscosity sensitivity of bimane molecular rotor-based fluorophores for imaging α-synuclein. J. Am. Chem. Soc. 147, 15115–15125 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Chen, M. Z. et al. A thiol probe for measuring unfolded protein load and proteostasis in cells. Nat. Commun. 8, 474 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  91. Wang, L. et al. Xanthone-based solvatochromic fluorophores for quantifying micropolarity of protein aggregates. Chem. Sci. 13, 12540–12549 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Zhang, T. et al. Near-infrared aggregation-induced emission luminogens for in vivo theranostics of Alzheimer’s disease. Angew. Chem. Int. Ed. 62, e202211550 (2023).

    Article  CAS  Google Scholar 

  93. Kubánková, M. et al. Probing supramolecular protein assembly using covalently attached fluorescent molecular rotors. Biomaterials 139, 195–201 (2017).

    Article  PubMed  Google Scholar 

  94. Huo, M. et al. Phosphorescent acyclic cucurbituril solid supramolecular multicolour delayed fluorescence behaviour. Chem. Sci. 15, 5163–5173 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Wang, Z. et al. A solvatochromic near infrared fluorophore sensitive to the full amyloid beta aggregation pathway. J. Am. Chem. Soc. 147, 18685–18693 (2025).

    Article  CAS  PubMed  Google Scholar 

  96. Wolstenholme, C. H. et al. AggFluor: fluorogenic toolbox enables direct visualization of the multi-step protein aggregation process in live cells. J. Am. Chem. Soc. 142, 17515–17523 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Zhang, C. et al. Ultrafast detection of monoamine oxidase A in live cells and clinical glioma tissues using an affinity binding-based two-photon fluorogenic probe. Angew. Chem. Int. Ed. 62, e202310134 (2023).

    Article  CAS  Google Scholar 

  98. Jiang, L. et al. EBNA1-targeted probe for the imaging and growth inhibition of tumours associated with the Epstein–Barr virus. Nat. Biomed. Eng. 1, 42 (2017).

    Article  CAS  Google Scholar 

  99. Wu, X. et al. A universal and programmable platform based on fluorescent peptide-conjugated probes for detection of proteins in organelles of living cells. Angew. Chem. Int. Ed. 136, e202400766 (2024).

    Article  Google Scholar 

  100. Shi, X. et al. A red-emissive antibody-AIEgen conjugate for turn-on and wash-free imaging of specific cancer cells. Chem. Sci. 8, 7014–7024 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Liu, J. et al. Near-infrared bioorthogonally activatable fluorescence probe for in vivo imaging of immune checkpoint in cancer. Adv. Funct. Mater. 35, 2508396 (2025).

    Article  CAS  Google Scholar 

  102. Zuo, S. et al. Rapid sorting and auxiliary evaluation of malignant breast tumors by accurate imaging analysis of metastasis-related biomarker. Sci. Adv. 11, eadr5541 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Xu, W., Yi, S., Liu, J., Jiang, Y. & Huang, J. Nitrile-aminothiol bioorthogonal near-infrared fluorogenic probes for ultrasensitive in vivo imaging. Nat. Commun. 16, 8 (2025).

    Article  PubMed  PubMed Central  Google Scholar 

  104. Feng, Y. et al. Activatable fluorescence/photoacoustic macromolecular probe for imaging of tumor-associated natural killer cells. Angew. Chem. Int. Ed. 64, e202507765 (2025).

    Article  CAS  Google Scholar 

  105. Luo, X., Hu, E., Deng, F., Zhang, C. & Xian, Y. A dual-enzyme activated fluorescent probe for precise identification of tumor senescence. Chem. Sci. 16, 6507–6514 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Shen, Y. et al. Dual-locked fluorescent probes activated by aminopeptidase N and the tumor redox environment for high-precision imaging of tumor boundaries. Angew. Chem. Int. Ed. 136, e202406332 (2024).

    Article  Google Scholar 

  107. Reja, S. I. et al. Controlling intramolecular rotation with five-membered heterocycles facilitates the design of highly cell-permeable xanthene-based fluorogenic probes. J. Am. Chem. Soc. 147, 47997–48012 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Reese, A. E. et al. Inserting “off-to-on” BODIPY tags into cytokines: a fluorogenic interleukin IL-33 for real-time imaging of immune cells. ACS Cent. Sci. 10, 143–154 (2024).

    Article  CAS  PubMed  Google Scholar 

  109. Bertolini, M. et al. Nonperturbative fluorogenic labeling of immunophilins enables the wash-free detection of immunosuppressants. ACS Cent. Sci. 10, 969–977 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Vahrmeijer, A. L., Hutteman, M., Van Der Vorst, J. R., Van De Velde, C. J. H. & Frangioni, J. V. Image-guided cancer surgery using near-infrared fluorescence. Nat. Rev. Clin. Oncol. 10, 507–518 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Wang, Q. et al. Unfolded protein-based sandwich AIE probe imparts high fluorescent contrast for pan-cancer surgical navigation. Anal. Chem. 96, 3609–3617 (2024).

    Article  CAS  PubMed  Google Scholar 

  112. Li, T. et al. Non-invasive in vivo monitoring of PROTAC-mediated protein degradation using an environment-sensitive reporter. Nat. Commun. 16, 1892 (2025).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Zhang, W. et al. A covalent self-reporting peptide degrader enables real-time monitoring of targeted protein degradation in vivo. J. Am. Chem. Soc. 147, 27862–27875 (2025).

    Article  CAS  PubMed  Google Scholar 

  114. Wang, K. et al. A nuclear-targeted AIE photosensitizer for enzyme inhibition and photosensitization in cancer cell ablation. Angew. Chem. Int. Ed. 61, e202114600 (2022).

    Article  CAS  Google Scholar 

  115. Huang, J. et al. Near-infrared chemiluminophore switches photodynamic processes via protein complexation for biomarker-activatable cancer therapy. Angew. Chem. Int. Ed. 64, e202421962 (2025).

    Article  CAS  Google Scholar 

  116. Qin, S. et al. Protein-confined rotor strategy for quantum yield enhancement in supramolecular photosensitizers toward sentinel lymph node-targeted photodynamic immunoactivation. ACS Nano 19, 24985–25006 (2025).

    Article  CAS  PubMed  Google Scholar 

  117. Zhang, L. et al. Transformable peptide nanoparticles arrest HER2 signalling and cause cancer cell death in vivo. Nat. Nanotechnol. 15, 145–153 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Yu, W.-T., Wu, T.-W., Huang, C.-L., Chen, I.-C. & Tan, K.-T. Protein sensing in living cells by molecular rotor-based fluorescence-switchable chemical probes. Chem. Sci. 7, 301–307 (2016).

    Article  CAS  PubMed  Google Scholar 

  119. Qin, Y. et al. CCNJ and DCNJ are bright julolidine-based fluorescent molecular rotors. Sens. Actuators B Chem. 444, 138472 (2025).

    Article  CAS  Google Scholar 

  120. Shi, L. et al. An activity-based photosensitizer to reverse hypoxia and oxidative resistance for tumor photodynamic eradication. Adv. Mater. 34, 2206659 (2022).

    Article  CAS  Google Scholar 

  121. Wang, W.-J. et al. Enzymatically catalyzed molecular aggregation. Nat. Commun. 15, 9999 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Zhang, Y. et al. Protein-triggered reassembly of quinocyanine nanosheets for intraoperative NIR-II cholangiography. Angew. Chem. Int. Ed. 65, e22772 (2025).

    Article  Google Scholar 

  123. Kühn, S. et al. SNAP-tag2 for faster and brighter protein labeling. Nat. Chem. Biol. 21, 1–8 (2025).

    Article  Google Scholar 

  124. Kompa, J. et al. Exchangeable HaloTag ligands for super-resolution fluorescence microscopy. J. Am. Chem. Soc. 145, 3075–3083 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Porzberg, N., Gries, K. & Johnsson, K. Exploiting covalent chemical labeling with self-labeling proteins. Annu. Rev. Biochem. 94, 29–58 (2025).

    Article  CAS  PubMed  Google Scholar 

  126. Marques, S. M. et al. Mechanism-based strategy for optimizing HaloTag protein labeling. JACS Au 2, 1324–1337 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. Fares, M. et al. A molecular rotor-based Halo-Tag ligand enables a fluorogenic proteome stress sensor to detect protein misfolding in mildly stressed proteome. Bioconjug. Chem. 29, 215–224 (2018).

    Article  CAS  PubMed  Google Scholar 

  128. Coïs, J. et al. Design of bright chemogenetic reporters based on the combined engineering of fluorogenic molecular rotors and of the HaloTag protein. Chem. Eur. J. 30, e202400641 (2024).

    Article  PubMed  Google Scholar 

  129. Bachollet, S. P. J. T., Addi, C., Pietrancosta, N., Mallet, J. & Dumat, B. Fluorogenic protein probes with red and near-infrared emission for genetically targeted imaging. Chem. Eur. J. 26, 14467–14473 (2020).

    Article  CAS  PubMed  Google Scholar 

  130. Cheng, Z. et al. Fluorescent amino acids as versatile building blocks for chemical biology. Nat. Rev. Chem. 4, 275–290 (2020).

    Article  CAS  PubMed  Google Scholar 

  131. Tan, Z. et al. Time-resolved fluorescent proteins expand fluorescent microscopy in temporal and spectral domains. Cell 188, 6987–7005 (2025).

    Article  CAS  PubMed  Google Scholar 

  132. Goh, W. L. et al. Molecular rotors as conditionally fluorescent labels for rapid detection of biomolecular interactions. J. Am. Chem. Soc. 136, 6159–6162 (2014).

    Article  CAS  PubMed  Google Scholar 

  133. Kuru, E. et al. Rapid discovery and evolution of nanosensors containing fluorogenic amino acids. Nat. Commun. 15, 7531 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Dou, J. et al. De novo design of a fluorescence-activating β-barrel. Nature 561, 485–491 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  135. Wilson, D. L. & Kool, E. T. Ultrafast oxime formation enables efficient fluorescence light-up measurement of DNA base excision. J. Am. Chem. Soc. 141, 19379–19388 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Dziuba, D., Jurkiewicz, P., Cebecauer, M., Hof, M. & Hocek, M. A rotational BODIPY nucleotide: an environment-sensitive fluorescence-lifetime probe for DNA interactions and applications in live-cell microscopy. Angew. Chem. Int. Ed. 128, 182–186 (2016).

    Article  Google Scholar 

  137. Güixens-Gallardo, P. & Hocek, M. Acetophenyl-thienyl-aniline-linked nucleotide for construction of solvatochromic fluorescence light-up DNA probes sensing protein-DNA interactions. Chem. Eur. J. 27, 7090–7093 (2021).

    Article  PubMed  Google Scholar 

  138. Zhuang, Y. et al. Facile, fast-responsive, and photostable imaging of telomerase activity in living cells with a fluorescence turn-on manner. Anal. Chem. 88, 3289–3294 (2016).

    Article  CAS  PubMed  Google Scholar 

  139. Zhuang, Y. et al. Construction of AIEgens-based bioprobe with two fluorescent signals for enhanced monitor of extracellular and intracellular telomerase activity. Anal. Chem. 89, 2073–2079 (2017).

    Article  CAS  PubMed  Google Scholar 

  140. Wu, X. et al. Aggregation-induced emission luminogens reveal cell cycle-dependent telomerase activity in cancer cells. Natl Sci. Rev. 8, nwaa306 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  141. Liu, R. et al. Precisely detecting the telomerase activities by an AIEgen probe with dual signal outputs after cell-cycle synchronization. Anal. Chem. 94, 4874–4880 (2022).

    Article  CAS  PubMed  Google Scholar 

  142. Jiang, L. et al. Large Stokes shift fluorescent RNAs for dual-emission fluorescence and bioluminescence imaging in live cells. Nat. Methods 20, 1563–1572 (2023).

    Article  CAS  PubMed  Google Scholar 

  143. Chen, Z. et al. Photoactivatable RNA tags for subcellular photolabeling of RNA. J. Am. Chem. Soc. 147, 31650–31661 (2025).

    Article  CAS  PubMed  Google Scholar 

  144. Wu, J. et al. Self-assembly of intracellular multivalent RNA complexes using dimeric corn and beetroot aptamers. J. Am. Chem. Soc. 144, 5471–5477 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. Xu, H. et al. Chemoproteomic profiling unveils binding and functional diversity of endogenous proteins that interact with endogenous triplex DNA. Nat. Chem. 16, 1811–1821 (2024).

    Article  CAS  PubMed  Google Scholar 

  146. Böttcher, T., Pitscheider, M. & Sieber, S. A. Natural products and their biological targets: proteomic and metabolomic labeling strategies. Angew. Chem. Int. Ed. 49, 2680–2698 (2010).

    Article  Google Scholar 

  147. Tan, X. et al. Chemical proteomics probes: classification, applications, and future perspectives in proteome-wide studies. Proteomics 25, 62–75 (2025).

    Article  CAS  PubMed  Google Scholar 

  148. Bi, Y. et al. Photocatalytic labelling-enabled subcellular-resolved RNA profiling and synchronous multi-omics investigation. Nat. Chem. 17, 1871–1882 (2025).

    Article  CAS  PubMed  Google Scholar 

  149. Shen, D. et al. Developing an affinity-based chemical proteomics method to in situ capture amorphous aggregated proteome and profile its heterogeneity in stressed cells. Anal. Chem. 95, 6358–6366 (2023).

    Article  CAS  PubMed  Google Scholar 

  150. Dong, X. et al. Integrated imaging and proteomic sensors resolve proteome aggregation in liver caused by non-steroidal anti-inflammatory drug overdose. ACS Sens. 8, 2247–2254 (2023).

    Article  CAS  PubMed  Google Scholar 

  151. Zhang, S. et al. Global analysis of endogenous protein disorder in cells. Nat. Methods 22, 124–134 (2025).

    Article  CAS  PubMed  Google Scholar 

  152. Xiang, J. et al. Chemical probe-enabled lipid droplet proteomics. J. Am. Chem. Soc. 147, 10724–10736 (2025).

    Article  CAS  PubMed  Google Scholar 

  153. Miki, T. et al. A conditional proteomics approach to identify proteins involved in zinc homeostasis. Nat. Methods 13, 931–937 (2016).

    Article  CAS  PubMed  Google Scholar 

  154. Feng, H. et al. Enabling photo-crosslinking and photo-sensitizing properties for synthetic fluorescent protein chromophores. Angew. Chem. Int. Ed. 135, e202215215 (2023).

    Article  Google Scholar 

  155. Zhai, Y. et al. Global profiling of functional histidines in live cells using small-molecule photosensitizer and chemical probe relay labelling. Nat. Chem. 16, 1546–1557 (2024).

    Article  CAS  PubMed  Google Scholar 

  156. Sun, R. et al. 1000 fold ultra-photosensitized fluorescent protein mimics toward photocatalytic proximity labeling and proteomic profiling functions. Adv. Sci. 12, 2413063 (2025).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported in part by the research works of the authors under the National Natural Science Foundation of China (22474131, U24A20502, 22504132), the National Key R&D Program of China (2021YFA1200403, 2025YFC2708402), the Natural Science Foundation of Hubei Province (2024AFA001, 2024AFB106), the Natural Science Foundation of Shenzhen (JCYJ20230807113706013), and the Guangdong Basic and Applied Basic Research Foundation (2023A1515110223).

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Author notes

  1. These authors contributed equally: Xia Wu, Yuxuan Hu.

Authors and Affiliations

  1. State Key Laboratory of Geomicrobiology and Environmental Changes, Faculty of Materials Science and Chemistry, China University of Geosciences, Wuhan, China

    Xia Wu, Quan Wang, Fan Xia & Xiaoding Lou

  2. School of Chemistry, Chemical Engineering and Biotechnology, Nanyang Technological University, Singapore, Singapore

    Yuxuan Hu & Kanyi Pu

  3. Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, Singapore

    Yuxuan Hu & Kanyi Pu

  4. State Key Laboratory of Natural and Biomimetic Drugs, Chemical Biology Center, Department of Molecular and Cellular Pharmacology, School of Pharmaceutical Sciences, Peking University, Beijing, China

    Wei Zhang & Tao Liu

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  1. Xia Wu
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  4. Wei Zhang
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  5. Tao Liu
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Contributions

K.P., X.L., F.X. and T.L. conceived the outline, designed the figures and edited the original draft. X.W., Y.H. and X.L. drew the figures and draft. Y.H., Q.W. and W.Z. helped to revise the paper. All of the authors contributed to writing the Review and approved the final version.

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Correspondence to Tao Liu, Fan Xia, Xiaoding Lou or Kanyi Pu.

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Nature Reviews Chemistry thanks Marc Vendrell and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Wu, X., Hu, Y., Wang, Q. et al. Molecular rotor-based probes for protein monitoring in biomedical research. Nat Rev Chem 10, 350–366 (2026). https://doi.org/10.1038/s41570-026-00822-x

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