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.
This is a preview of subscription content, access via your institution
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 per month
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Get just this article for as long as you need it
Prices may be subject to local taxes which are calculated during checkout
Source data are provided with this paper. All other data are available from the corresponding author.
Perrin, R. J., Fagan, A. M. & Holtzman, D. M. Multimodal techniques for diagnosis and prognosis of Alzheimer’s disease. Nature 461, 916–922 (2009).
Selkoe, D. J. & Hardy, J. The amyloid hypothesis of Alzheimer’s disease at 25 years. EMBO Mol. Med. 8, 595–608 (2016).
Gremer, L. et al. Fibril structure of amyloid-β(1-42) by cryo-electron microscopy. Science 358, 116–119 (2017).
Ross, C. A. & Poirier, M. A. Protein aggregation and neurodegenerative disease. Nat. Med. 10, S10–S17 (2004).
Zhou, J. et al. Fluorescent diagnostic probes in neurodegenerative diseases. Adv. Mater. 32, e2001945 (2020).
Aliyan, A., Cook, N. P. & Marti, A. A. Interrogating amyloid aggregates using fluorescent probes. Chem. Rev. 119, 11819–11856 (2019).
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).
Sun, X. et al. The mechanisms of boronate ester formation and fluorescent turn-on in ortho-aminomethylphenylboronic acids. Nat. Chem. 11, 768–778 (2019).
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).
Xu, H. et al. Analyte regeneration fluorescent probes for formaldehyde enabled by regiospecific formaldehyde-induced intramolecularity. J. Am. Chem. Soc. 140, 16408–16412 (2018).
Chan, J., Dodani, S. C. & Chang, C. J. Reaction-based small-molecule fluorescent probes for chemoselective bioimaging. Nat. Chem. 4, 973–984 (2012).
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).
Han, H. H. et al. Small-molecule fluorescence-based probes for interrogating major organ diseases. Chem. Soc. Rev. 50, 9391–9429 (2021).
Jun, Y. W. et al. Frontiers in probing Alzheimer’s disease biomarkers with fluorescent small molecules. ACS Cent. Sci. 5, 209–217 (2019).
Vassar, P. S. & Culling, C. F. Fluorescent stains, with special reference to amyloid and connective tissues. Arch. Pathol. 68, 487–498 (1959).
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).
Biancalana, M. & Koide, S. Molecular mechanism of Thioflavin-T binding to amyloid fibrils. Biochim. Biophys. Acta 1804, 1405–1412 (2010).
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).
Luo, J. et al. Aggregation-induced emission of 1-methyl-1,2,3,4,5-pentaphenylsilole. Chem. Commun. (Camb.) 2001, 1740–1741 (2001).
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).
Ding, D., Li, K., Liu, B. & Tang, B. Z. Bioprobes based on AIE fluorogens. Acc. Chem. Res. 46, 2441–2453 (2013).
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).
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).
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).
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).
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).
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).
Wu, D. et al. Fluorescent chemosensors: the past, present and future. Chem. Soc. Rev. 46, 7105–7123 (2017).
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).
Yu, Q. et al. Semisynthetic sensor proteins enable metabolic assays at the point of care. Science 361, 1122–1126 (2018).
Zhu, H. et al. Synthesis of an ultrasensitive BODIPY-derived fluorescent probe for detecting HOCl in live cells. Nat. Protoc. 13, 2348–2361 (2018).
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).
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).
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).
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).
Li, H. et al. Activity-based smart AIEgens for detection, bioimaging, and therapeutics: recent progress and outlook. Aggregate 2, 51 (2021).
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).
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).
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).
Cao, K. J. & Yang, J. Translational opportunities for amyloid-targeting fluorophores. Chem. Commun. 54, 9107–9118 (2018).
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).
Liu, X., Duan, Y. & Liu, B. Nanoparticles as contrast agents for photoacoustic brain imaging. Aggregate 2, 4–19 (2021).
Li, G. & Li, Y. M. Modulating the aggregation of amyloid proteins by macrocycles. Aggregate 3, 161 (2022).
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).
Hong, G., Antaris, A. L. & Dai, H. Near-infrared fluorophores for biomedical imaging. Nat. Biomed. Eng. 1, 1–22 (2017).
Wang, S., Li, B. & Zhang, F. Molecular fluorophores for deep-tissue bioimaging. ACS Cent. Sci. 6, 1302–1316 (2020).
Kostelnik, T. I. & Orvig, C. Radioactive main group and rare earth metals for imaging and therapy. Chem. Rev. 119, 902–956 (2019).
Price, E. W. & Orvig, C. Matching chelators to radiometals for radiopharmaceuticals. Chem. Soc. Rev. 43, 260–290 (2014).
Tao, Y. et al. Sequence-activated fluorescent nanotheranostics for real-time profiling pancreatic cancer. JACS Au 2, 246–257 (2022).
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).
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).
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).
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).
Heo, C. H. et al. A two-photon fluorescent probe for amyloid-β plaques in living mice. Chem. Commun. 49, 1303–1305 (2013).
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).
Teoh, C. L. et al. Chemical fluorescent probe for detection of Aβ oligomers. J. Am. Chem. Soc. 137, 13503–13509 (2015).
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).
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).
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).
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).
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).
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).
Z.G. has filed a patent application for the QM-FN-SO3 probe. The patent application number is CN201811069366 (patent number: ZL 201811069366.9).
Peer review information
Nature Protocols thanks Juyoung Yoon and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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 Figs. 1–9 and Methods 1 and 2
Supplementary Data 1
Statistical data for Supplementary Figs. 2, 3, 7b and 9a,b
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
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 (2023). https://doi.org/10.1038/s41596-022-00789-1
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.