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Preparation of near-infrared AIEgen-active fluorescent probes for mapping amyloid-β plaques in brain tissues and living mice

Nature Protocols volume 18, pages 1316–1336 (2023)Cite this article

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Abstract

Fibrillar aggregates of the amyloid-β protein (Aβ) are the main component of the senile plaques found in brains of patients with Alzheimer’s disease (AD). Development of probes allowing the noninvasive and high-fidelity mapping of Aβ plaques in vivo is critical for AD early detection, drug screening and biomedical research. QM-FN-SO3 (quinoline-malononitrile-thiophene-(dimethylamino)phenylsulfonate) is a near-infrared aggregation-induced-emission-active fluorescent probe capable of crossing the blood–brain barrier (BBB) and ultrasensitively lighting up Aβ plaques in living mice. Herein, we describe detailed procedures for the two-stage synthesis of QM-FN-SO3 and its applications for mapping Aβ plaques in brain tissues and living mice. Compared with commercial thioflavin (Th) derivatives ThT and ThS (the gold standard for detection of Aβ aggregates) and other reported Aβ plaque fluorescent probes, QM-FN-SO3 confers several advantages, such as long emission wavelength, large Stokes shift, ultrahigh sensitivity, good BBB penetrability and miscibility in aqueous biological media. The preparation of QM-FN-SO3 takes ~2 d, and the confocal imaging experiments for Aβ plaque visualization, including the preparation for mouse brain sections, take ~7 d. Notably, acquisition and analyses for in vivo visualization of Aβ plaques in mice can be completed within 1 h and require only a basic knowledge of spectroscopy and chemistry.

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Fig. 1: Comparison of QM-FN-SO3 with extensively used ThT for sensing Aβ aggregates.
Fig. 2: Step-by-step strategy for building up probes enabling ultrasensitive in vivo mapping of Aβ plaques.
Fig. 3: Overview of applying QM-FN-SO3 for brain section and animal experiments.
Fig. 4: Spectroscopic characterization of QM-FN-SO3 toward Aβ aggregates.
Fig. 5: High-fidelity mapping of Aβ plaques in brain tissues and validation of the BBB penetrability of QM-FN-SO3.
Fig. 6: In vivo mapping of Aβ plaques in AD model mice.

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Source data are provided with this paper. All other data are available from the corresponding author.

References

  1. Perrin, R. J., Fagan, A. M. & Holtzman, D. M. Multimodal techniques for diagnosis and prognosis of Alzheimer’s disease. Nature 461, 916–922 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Selkoe, D. J. & Hardy, J. The amyloid hypothesis of Alzheimer’s disease at 25 years. EMBO Mol. Med. 8, 595–608 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Gremer, L. et al. Fibril structure of amyloid-β(1-42) by cryo-electron microscopy. Science 358, 116–119 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  PubMed  Google Scholar 

  5. Zhou, J. et al. Fluorescent diagnostic probes in neurodegenerative diseases. Adv. Mater. 32, e2001945 (2020).

    Article  PubMed  Google Scholar 

  6. Aliyan, A., Cook, N. P. & Marti, A. A. Interrogating amyloid aggregates using fluorescent probes. Chem. Rev. 119, 11819–11856 (2019).

    Article  CAS  PubMed  Google Scholar 

  7. Yin, J. et al. Preparation of a cyanine-based fluorescent probe for highly selective detection of glutathione and its use in living cells and tissues of mice. Nat. Protoc. 10, 1742–1754 (2015).

    Article  CAS  PubMed  Google Scholar 

  8. Sun, X. et al. The mechanisms of boronate ester formation and fluorescent turn-on in ortho-aminomethylphenylboronic acids. Nat. Chem. 11, 768–778 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Long, L. et al. A mitochondria-specific fluorescent probe for visualizing endogenous hydrogen cyanide fluctuations in neurons. J. Am. Chem. Soc. 140, 1870–1875 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Xu, H. et al. Analyte regeneration fluorescent probes for formaldehyde enabled by regiospecific formaldehyde-induced intramolecularity. J. Am. Chem. Soc. 140, 16408–16412 (2018).

    Article  CAS  PubMed  Google Scholar 

  11. 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 

  12. Tong, H., Lou, K. & Wang, W. Near-infrared fluorescent probes for imaging of amyloid plaques in Alzheimer’s disease. Acta Pharm. Sin. B 5, 25–33 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Han, H. H. et al. Small-molecule fluorescence-based probes for interrogating major organ diseases. Chem. Soc. Rev. 50, 9391–9429 (2021).

    Article  CAS  PubMed  Google Scholar 

  14. Jun, Y. W. et al. Frontiers in probing Alzheimer’s disease biomarkers with fluorescent small molecules. ACS Cent. Sci. 5, 209–217 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Vassar, P. S. & Culling, C. F. Fluorescent stains, with special reference to amyloid and connective tissues. Arch. Pathol. 68, 487–498 (1959).

    CAS  PubMed  Google Scholar 

  16. Bacskai, B. J. et al. Imaging of amyloid-β deposits in brains of living mice permits direct observation of clearance of plaques with immunotherapy. Nat. Med. 7, 369–372 (2001).

    Article  CAS  PubMed  Google Scholar 

  17. Biancalana, M. & Koide, S. Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochim. Biophys. Acta 1804, 1405–1412 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Amdursky, N., Erez, Y. & Huppert, D. Molecular rotors: what lies behind the high sensitivity of the thioflavin-T fluorescent marker. Acc. Chem. Res. 45, 1548–1557 (2012).

    Article  CAS  PubMed  Google Scholar 

  19. Luo, J. et al. Aggregation-induced emission of 1-methyl-1,2,3,4,5-pentaphenylsilole. Chem. Commun. (Camb.) 2001, 1740–1741 (2001).

    Article  Google Scholar 

  20. Mei, J., Leung, N. L., Kwok, R. T., Lam, J. W. & Tang, B. Z. Aggregation-induced emission: together we shine, united we soar! Chem. Rev. 115, 11718–11940 (2015).

    Article  CAS  PubMed  Google Scholar 

  21. Ding, D., Li, K., Liu, B. & Tang, B. Z. Bioprobes based on AIE fluorogens. Acc. Chem. Res. 46, 2441–2453 (2013).

    Article  CAS  PubMed  Google Scholar 

  22. Guo, Z., Yan, C. & Zhu, W. H. High-performance quinoline-malononitrile core as a building block for the diversity-oriented synthesis of AIEgens. Angew. Chem. Int. Ed. Engl. 59, 9812–9825 (2020).

    Article  CAS  PubMed  Google Scholar 

  23. Fu, W. et al. Rational design of near-infrared aggregation-induced-emission-active probes: in situ mapping of amyloid-β plaques with ultrasensitivity and high-fidelity. J. Am. Chem. Soc. 141, 3171–3177 (2019).

    Article  CAS  PubMed  Google Scholar 

  24. Shao, A. et al. Far-red and near-IR AIE-active fluorescent organic nanoprobes with enhanced tumor-targeting efficacy: shape-specific effects. Angew. Chem. Int. Ed. Engl. 54, 7275–7280 (2015).

    Article  CAS  PubMed  Google Scholar 

  25. Zhang, Y. et al. A sequential dual-lock strategy for photoactivatable chemiluminescent probes enabling bright duplex optical imaging. Angew. Chem. Int. Ed. Engl. 59, 9059–9066 (2020).

    Article  CAS  PubMed  Google Scholar 

  26. Gu, K. et al. An enzyme-activatable probe liberating AIEgens: on-site sensing and long-term tracking of β-galactosidase in ovarian cancer cells. Chem. Sci. 10, 398–405 (2019).

    Article  CAS  PubMed  Google Scholar 

  27. Shao, A. D. et al. Insight into aggregation-induced emission characteristics of red-emissive quinoline-malononitrile by cell tracking and real-time trypsin detection. Chem. Sci. 5, 1383–1389 (2014).

    Article  CAS  Google Scholar 

  28. Wu, D. et al. Fluorescent chemosensors: the past, present and future. Chem. Soc. Rev. 46, 7105–7123 (2017).

    Article  CAS  PubMed  Google Scholar 

  29. McMahon, B. K. & Gunnlaugsson, T. Selective detection of the reduced form of glutathione (GSH) over the oxidized (GSSG) form using a combination of glutathione reductase and a Tb(III)-cyclen maleimide based lanthanide luminescent ‘switch on’ assay. J. Am. Chem. Soc. 134, 10725–10728 (2012).

    Article  CAS  PubMed  Google Scholar 

  30. Yu, Q. et al. Semisynthetic sensor proteins enable metabolic assays at the point of care. Science 361, 1122–1126 (2018).

    Article  CAS  PubMed  Google Scholar 

  31. Zhu, H. et al. Synthesis of an ultrasensitive BODIPY-derived fluorescent probe for detecting HOCl in live cells. Nat. Protoc. 13, 2348–2361 (2018).

    Article  CAS  PubMed  Google Scholar 

  32. Li, H. et al. Ferroptosis accompanied by •OH generation and cytoplasmic viscosity increase revealed via dual-functional fluorescence probe. J. Am. Chem. Soc. 141, 18301–18307 (2019).

    Article  CAS  PubMed  Google Scholar 

  33. Li, H. et al. An activatable AIEgen probe for high-fidelity monitoring of overexpressed tumor enzyme activity and its application to surgical tumor excision. Angew. Chem. Int. Ed. Engl. 59, 10186–10195 (2020).

    Article  CAS  PubMed  Google Scholar 

  34. 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 

  35. Ren, T. B. et al. A general strategy for development of activatable NIR-II fluorescent probes for in vivo high-contrast bioimaging. Angew. Chem. Int. Ed. Engl. 60, 800–805 (2021).

    Article  CAS  PubMed  Google Scholar 

  36. Li, H. et al. Activity-based smart AIEgens for detection, bioimaging, and therapeutics: recent progress and outlook. Aggregate 2, 51 (2021).

    Google Scholar 

  37. Tang, Y., Zhao, Y. & Lin, W. Preparation of robust fluorescent probes for tracking endogenous formaldehyde in living cells and mouse tissue slices. Nat. Protoc. 15, 3499–3526 (2020).

    Article  CAS  PubMed  Google Scholar 

  38. 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 

  39. Zhang, D. et al. Naked-eye readout of analyte-induced NIR fluorescence responses by an initiation-input-transduction nanoplatform. Angew. Chem. Int. Ed. Engl. 59, 695–699 (2020).

    Article  PubMed  Google Scholar 

  40. Cao, K. J. & Yang, J. Translational opportunities for amyloid-targeting fluorophores. Chem. Commun. 54, 9107–9118 (2018).

    Article  CAS  Google Scholar 

  41. Staderini, M., Martin, M. A., Bolognesi, M. L. & Menendez, J. C. Imaging of β-amyloid plaques by near infrared fluorescent tracers: a new frontier for chemical neuroscience. Chem. Soc. Rev. 44, 1807–1819 (2015).

    Article  CAS  PubMed  Google Scholar 

  42. Liu, X., Duan, Y. & Liu, B. Nanoparticles as contrast agents for photoacoustic brain imaging. Aggregate 2, 4–19 (2021).

    Article  CAS  Google Scholar 

  43. Li, G. & Li, Y. M. Modulating the aggregation of amyloid proteins by macrocycles. Aggregate 3, 161 (2022).

    Google Scholar 

  44. Padhi, D. & Govindaraju, T. Mechanistic insights for drug repurposing and the design of hybrid drugs for Alzheimer’s disease. J. Med. Chem. 65, 7088–7105 (2022).

    Article  CAS  PubMed  Google Scholar 

  45. Hong, G., Antaris, A. L. & Dai, H. Near-infrared fluorophores for biomedical imaging. Nat. Biomed. Eng. 1, 1–22 (2017).

    Article  Google Scholar 

  46. Wang, S., Li, B. & Zhang, F. Molecular fluorophores for deep-tissue bioimaging. ACS Cent. Sci. 6, 1302–1316 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Kostelnik, T. I. & Orvig, C. Radioactive main group and rare earth metals for imaging and therapy. Chem. Rev. 119, 902–956 (2019).

    Article  CAS  PubMed  Google Scholar 

  48. Price, E. W. & Orvig, C. Matching chelators to radiometals for radiopharmaceuticals. Chem. Soc. Rev. 43, 260–290 (2014).

    Article  CAS  PubMed  Google Scholar 

  49. Tao, Y. et al. Sequence-activated fluorescent nanotheranostics for real-time profiling pancreatic cancer. JACS Au 2, 246–257 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Hintersteiner, M. et al. In vivo detection of amyloid-β deposits by near-infrared imaging using an oxazine-derivative probe. Nat. Biotechnol. 23, 577–583 (2005).

    Article  CAS  PubMed  Google Scholar 

  51. Aslund, A. et al. Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral amyloidoses. ACS Chem. Biol. 4, 673–684 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Berg, I., Nilsson, K. P., Thor, S. & Hammarstrom, P. Efficient imaging of amyloid deposits in Drosophila models of human amyloidoses. Nat. Protoc. 5, 935–944 (2010).

    Article  CAS  PubMed  Google Scholar 

  53. Cao, K. et al. Aminonaphthalene 2-cyanoacrylate (ANCA) probes fluorescently discriminate between amyloid-β and prion plaques in brain. J. Am. Chem. Soc. 134, 17338–17341 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Heo, C. H. et al. A two-photon fluorescent probe for amyloid-β plaques in living mice. Chem. Commun. 49, 1303–1305 (2013).

    Article  CAS  Google Scholar 

  55. Shin, J. et al. Harnessing intramolecular rotation to enhance two-photon imaging of Aβ plaques through minimizing background fluorescence. Angew. Chem. Int. Ed. Engl. 58, 5648–5652 (2019).

    Article  CAS  PubMed  Google Scholar 

  56. Teoh, C. L. et al. Chemical fluorescent probe for detection of Aβ oligomers. J. Am. Chem. Soc. 137, 13503–13509 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Ran, C. et al. Design, synthesis, and testing of difluoroboron-derivatized curcumins as near-infrared probes for in vivo detection of amyloid-β deposits. J. Am. Chem. Soc. 131, 15257–15261 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Zhang, X. et al. Design and synthesis of curcumin analogues for in vivo fluorescence imaging and inhibiting copper-induced cross-linking of amyloid β species in Alzheimer’s disease. J. Am. Chem. Soc. 135, 16397–16409 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Cui, M. et al. Smart near-infrared fluorescence probes with donor-acceptor structure for in vivo detection of β-amyloid deposits. J. Am. Chem. Soc. 136, 3388–3394 (2014).

    Article  CAS  PubMed  Google Scholar 

  60. Fu, H. et al. Highly sensitive near-infrared fluorophores for in vivo detection of amyloid-β plaques in Alzheimer’s disease. J. Med. Chem. 58, 6972–6983 (2015).

    Article  CAS  PubMed  Google Scholar 

  61. Kim, D. et al. Two-photon absorbing dyes with minimal autofluorescence in tissue imaging: application to in vivo imaging of amyloid-β plaques with a negligible background signal. J. Am. Chem. Soc. 137, 6781–6789 (2015).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by NSFC/China (22225805, 21878087 and 21908060), National Key Research and Development Program (2021YFA0910000), Innovation Program of Shanghai Municipal Education Commission, Shanghai Frontier Science Research Base of Optogenetic Techniques for Cell Metabolism (Shanghai Municipal Education Commission, grant 2021 Sci & Tech 03-28), Shanghai Municipal Science and Technology Major Project (Grant 2018SHZDZX03), Shanghai Science and Technology Committee Rising-Star Program (22QC1400400) and Programme of Introducing Talents of Discipline to Universities (B16017).

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Authors and Affiliations

  1. Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine Chemicals, Frontiers Science Center for Materiobiology and Dynamic Chemistry, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai, China

    Chenxu Yan, Jianfeng Dai, Yongkang Yao, Wei Fu, He Tian, Wei-Hong Zhu & Zhiqian Guo

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Contributions

All the experiments were conducted by C.Y., J.D., Y.Y. and W.F. with the supervision of H.T., W.-H.Z. and Z.G. All the authors analyzed the data and contributed to the manuscript writing.

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Correspondence to Zhiqian Guo.

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Z.G. has filed a patent application for the QM-FN-SO3 probe. The patent application number is CN201811069366 (patent number: ZL 201811069366.9).

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Nature Protocols thanks Juyoung Yoon and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Fu, W. et al. J. Am. Chem. Soc. 141, 3171–3177 (2019): https://doi.org/10.1021/jacs.8b12820

Supplementary information

Supplementary Information

Supplementary Figs. 1–9 and Methods 1 and 2

Supplementary Data 1

Statistical data for Supplementary Figs. 2, 3, 7b and 9a,b

Source data

Source Data Fig. 2

Value of S/N ratio of the probes (ThT, DCM-N, QM-FN and QM-FN-SO3)

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Yan, C., Dai, J., Yao, Y. et al. Preparation of near-infrared AIEgen-active fluorescent probes for mapping amyloid-β plaques in brain tissues and living mice. Nat Protoc 18, 1316–1336 (2023). https://doi.org/10.1038/s41596-022-00789-1

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