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Cited by 123Alzheimer's disease research and treatmentsMolecular Sensors and Ion DetectionLuminescence and Fluorescent Materials

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Turn-on chemiluminescence probes and dual-amplification of signal for detection of amyloid beta species in vivo

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Yang, Jing, Yin, Wei, Van, Richard, Yin, Keyi, Wang, Peng, Zheng, Chao, Zhu, Biyue, Ran, Kathleen, Zhang, Can, Kumar, Mohanraja, Shao, Yihan, Ran, Chongzhao·2020·Nature Communications
DOI 10.1038/s41467-020-17783-4PMC PMC7426431

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Abstract

Turn-on fluorescence imaging is routinely studied; however, turn-on chemiluminescence has been rarely explored for in vivo imaging. Herein, we report the design and validation of chemiluminescence probe ADLumin-1 as a turn-on probe for amyloid beta (Aβ) species. Two-photon imaging indicates that ADLumin-1 can efficiently cross the blood–brain barrier and provides excellent contrast for Aβ plaques and cerebral amyloid angiopathy. In vivo brain imaging shows that the chemiluminescence signal of ADLumin-1 from 5-month-old transgenic 5xFAD mice is 1.80-fold higher than that from the age-matched wild-type mice. Moreover, we demonstrate that it is feasible to further d ually- a mplify s ignal via c hemiluminescence r esonance e nergy t ransfer (DAS-CRET) using two non-conjugated smart probes (ADLumin-1 and CRANAD-3) in solutions, brain homogenates, and in vivo whole brain imaging. Our results show that DAS-CRET can provide a 2.25-fold margin between 5-month-old 5xFAD mice and wild type mice. We believe that our strategy could be extended to other aggregating-prone proteins.

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Fig. 1 Design and synthesis of ADLumin-1. a Diagram of designing of chemiluminescent probes that contain moiety A for generating chemiluminescence (green) and moiety B for binding to Aβs; b Chemical structures of the designed probes ADLumin-X (X = −1 and −2) and diagram for illustrating turn-on with ADLumin-X in the presence of Aβs; c) Synthetic route of ADLumin-Xs. Reagents and conditions: (I) TMP, n-BuLi, THF; (II) 5-bromo-2-aminopyrazine, Pd(PPh3)4, NaCO3 aq, 1,4-dioxane; (III) Methylglyoxal 1,1-dimethyl acetal, EtOH, HCl aq.
Fig. 1 Design and synthesis of ADLumin-1. a Diagram of designing of chemiluminescent probes that contain moiety A for generating chemiluminescence (green) and moiety B for binding to Aβs; b Chemical structures of the designed probes ADLumin-X (X = −1 and −2) and diagram for illustrating turn-on with ADLumin-X in the presence of Aβs; c) Synthetic route of ADLumin-Xs. Reagents and conditions: (I) TMP, n-BuLi, THF; (II) 5-bromo-2-aminopyrazine, Pd(PPh3)4, NaCO3 aq, 1,4-dioxane; (III) Methylglyoxal 1,1-dimethyl acetal, EtOH, HCl aq.CC BY · PubMed Central
Fig. 2 In vitro characterization of chemiluminescence properties of ADLumin-1. a Chemiluminescence spectrum of ADLumin-1 in DMSO; b Quantitative analysis of chemiluminescence intensity of ADLumin-1 in DMSO solutions before and after stirring. Representative images were inserted. c Quantification of chemiluminescence intensity of ADLumin-1 with (red) or without (black) bubbled oxygen; d LC-MS spectra of ADLumin-1 and ADLumin-3 (the oxidation product). Note: ADLumin-1 (t = 3.05 min), ADLumin-3 (t = 3.85 min); e Proposed oxygen-dependent mechanism of ADLumin-1 for chemiluminescence generation. Data are represented as mean ± s.d. with n = 3 biologically independent samples. P = 0.0009 (b). The P-values were generated by Graphpad Prism 8 with two-tailed unpaired t-test; ***P-value < 0.001; Source data underlying b and c are provided as Source Data file.
Fig. 2 In vitro characterization of chemiluminescence properties of ADLumin-1. a Chemiluminescence spectrum of ADLumin-1 in DMSO; b Quantitative analysis of chemiluminescence intensity of ADLumin-1 in DMSO solutions before and after stirring. Representative images were inserted. c Quantification of chemiluminescence intensity of ADLumin-1 with (red) or without (black) bubbled oxygen; d LC-MS spectra of ADLumin-1 and ADLumin-3 (the oxidation product). Note: ADLumin-1 (t = 3.05 min), ADLumin-3 (t = 3.85 min); e Proposed oxygen-dependent mechanism of ADLumin-1 for chemiluminescence generation. Data are represented as mean ± s.d. with n = 3 biologically independent samples. P = 0.0009 (b). The P-values were generated by Graphpad Prism 8 with two-tailed unpaired t-test; ***P-value < 0.001; Source data underlying b and c are provided as Source Data file.CC BY · PubMed Central
Fig. 3 In vitro characterization of fluorescence and chemiluminescence properties of ADLumin-1 in the presence of Aβs. a Fluorescence spectra of ADLumin-1 alone (red) and with Aβ40 aggregates (black); b Chemiluminescence spectra of ADLumin-1 alone (red) and with Aβ40 aggregates (black); c Molecule docking of ADLumin-1 (blue) with Aβ fibrils (PDB:5OQV). d In vitro chemiluminescence images of ADLumin-1 alone and with Aβ40 aggregates in PBS. e Quantification of chemiluminescence intensity in d. f In vitro chemiluminescence images of ADLumin-1 alone and with Aβ40 aggregates in mouse brain homogenate; g Quantification of chemiluminescence intensity in f. Data are represented as mean ± s.d. with n = 3 biologically independent samples. P = 0.0005 (e), P < 0.0001 (g). The P-values were generated by Graphpad Prism 8 with two-tailed unpaired t-test; ***P-value < 0.001, ****P-value < 0.0001. Source data underlying e and g are provided as Source Data file.
Fig. 3 In vitro characterization of fluorescence and chemiluminescence properties of ADLumin-1 in the presence of Aβs. a Fluorescence spectra of ADLumin-1 alone (red) and with Aβ40 aggregates (black); b Chemiluminescence spectra of ADLumin-1 alone (red) and with Aβ40 aggregates (black); c Molecule docking of ADLumin-1 (blue) with Aβ fibrils (PDB:5OQV). d In vitro chemiluminescence images of ADLumin-1 alone and with Aβ40 aggregates in PBS. e Quantification of chemiluminescence intensity in d. f In vitro chemiluminescence images of ADLumin-1 alone and with Aβ40 aggregates in mouse brain homogenate; g Quantification of chemiluminescence intensity in f. Data are represented as mean ± s.d. with n = 3 biologically independent samples. P = 0.0005 (e), P < 0.0001 (g). The P-values were generated by Graphpad Prism 8 with two-tailed unpaired t-test; ***P-value < 0.001, ****P-value < 0.0001. Source data underlying e and g are provided as Source Data file.CC BY · PubMed Central
Fig. 4 Chemiluminescence light penetration of ADLumin-1. a Chemiluminescence spectra of ADLumin-1 (black) and bioluminescence spectra of Luciferin (red); b Light penetration study with Luciferin and ADLumin-1. A glass tube filled with a solution of firefly luciferin with luciferase or ADLumin-1 with similar intensities before putting under the abdomen of two nude mice; c Images that were captured from the dorsal side with an IVIS imaging system after placing the tubes; d The quantification of the penetrated light in c. Nearly 5% light penetrated the whole body in both groups.
Fig. 4 Chemiluminescence light penetration of ADLumin-1. a Chemiluminescence spectra of ADLumin-1 (black) and bioluminescence spectra of Luciferin (red); b Light penetration study with Luciferin and ADLumin-1. A glass tube filled with a solution of firefly luciferin with luciferase or ADLumin-1 with similar intensities before putting under the abdomen of two nude mice; c Images that were captured from the dorsal side with an IVIS imaging system after placing the tubes; d The quantification of the penetrated light in c. Nearly 5% light penetrated the whole body in both groups.CC BY · PubMed Central
Fig. 5 In vitro and in vivo plaques imaging with ADLumin-1. a Representative brain slice image of cortex area of a 24-mo-old APP/PS1 mouse with ADLumin-1 staining; b Two-photon microscopic image of brain cortex area with ADLumin-1 in an alive 15-mo-old 5xFAD mouse. The blood vessels were highlighted by Rhodamine B isothiocyanate-Dextran, and the presentative image is merged images from two concurrent imaging channels. The yellow arrows indicate plaques, and the white arrows indicate CAA labeling; Scale bar: 50 μm. c Ex vivo histology of a mouse brain slice obtained after the two-photon imaging with ADLumin-1. d–f Quantitative analysis of SNR of plaques of in vitro slice (d) (mean ± s.d., n = 6 independent measurements), in vivo plaques (mean ± s.d., n = 5 independent measurements) and CAAs (mean ± s.d., n = 5 independent measurements) (e), and ex vivo plaques (mean ± s.d., n = 6 independent measurements) (f). P < 0.0001 (d); P < 0.0001 (e); and P < 0.0001 (f). The P-values were generated by Graphpad Prism 8 with two-tailed unpaired t-test; ***P-value < 0.001, ****P-value < 0.0001. Bkg background, CAA cerebral amyloid angiopathy. Source data underlying d–f are provided as Source Data file.
Fig. 5 In vitro and in vivo plaques imaging with ADLumin-1. a Representative brain slice image of cortex area of a 24-mo-old APP/PS1 mouse with ADLumin-1 staining; b Two-photon microscopic image of brain cortex area with ADLumin-1 in an alive 15-mo-old 5xFAD mouse. The blood vessels were highlighted by Rhodamine B isothiocyanate-Dextran, and the presentative image is merged images from two concurrent imaging channels. The yellow arrows indicate plaques, and the white arrows indicate CAA labeling; Scale bar: 50 μm. c Ex vivo histology of a mouse brain slice obtained after the two-photon imaging with ADLumin-1. d–f Quantitative analysis of SNR of plaques of in vitro slice (d) (mean ± s.d., n = 6 independent measurements), in vivo plaques (mean ± s.d., n = 5 independent measurements) and CAAs (mean ± s.d., n = 5 independent measurements) (e), and ex vivo plaques (mean ± s.d., n = 6 independent measurements) (f). P < 0.0001 (d); P < 0.0001 (e); and P < 0.0001 (f). The P-values were generated by Graphpad Prism 8 with two-tailed unpaired t-test; ***P-value < 0.001, ****P-value < 0.0001. Bkg background, CAA cerebral amyloid angiopathy. Source data underlying d–f are provided as Source Data file.CC BY · PubMed Central
Fig. 6 In vivo chemiluminescence imaging with ADLumin-1. a In vivo brain imaging of WT and 5XFAD mice with iv injection of ADLumin-1; b Quantitative analysis of the images in a; c Representative images of eyes in WT and 5xFAD mice after iv injection with ADLumin-1; d Quantitative analysis of the images in c (n = 4). Error bar: Stdev; Data points with its error bar stands for mean ± s.d. derived from n = 4 biologically independent animals. P < 0.0001 (b); P = 0.0098 (d). The P-values were generated by Graphpad Prism 8 with two-tailed unpaired t-test; ***P-value < 0.001; **P-value < 0.01. AD Alzheimer’s disease, WT wild-type. Source data underlying b and d are provided as Source Data file.
Fig. 6 In vivo chemiluminescence imaging with ADLumin-1. a In vivo brain imaging of WT and 5XFAD mice with iv injection of ADLumin-1; b Quantitative analysis of the images in a; c Representative images of eyes in WT and 5xFAD mice after iv injection with ADLumin-1; d Quantitative analysis of the images in c (n = 4). Error bar: Stdev; Data points with its error bar stands for mean ± s.d. derived from n = 4 biologically independent animals. P < 0.0001 (b); P = 0.0098 (d). The P-values were generated by Graphpad Prism 8 with two-tailed unpaired t-test; ***P-value < 0.001; **P-value < 0.01. AD Alzheimer’s disease, WT wild-type. Source data underlying b and d are provided as Source Data file.CC BY · PubMed Central
Fig. 7 In vitro validation of CRET with ADLumin-1/CRANAD-3 pair. a Proposed CRET model between ADLumin-1 and CRANAD-3 with Aβ40 fibrils. Two nonconjugated molecules upon binding to fibrils to bring ADLumin-1 (Donor) close enough to CRANAD-3 (Acceptor) to initiate CRET; b Spectrum of the CRET pair with Aβ40 fibrils in PBS (red line), and the peak was consistent with the emission of CRANAD-3 in the presence of Aβ40 fibrils. Chemiluminescence spectrum of ADLumin-1 with Aβ40 fibrils (black line); and spectrum of the mixture of ADLumin-1 and CRANAD-3 without Aβ40 fibrils (pink line). The FRETing efficiency was very high, evident by the low intensity at 500–560 nm range; c Spectrum of the CRET pair in brain homogenate (red line), evident by a decrease in ADLumin-1 emission (black line) and increase in CRANAD-3 emission; d Spectral unmixing of DAS-CRET to separate the contribution from ADLumin-1 only (Unmixed #1), and CRET (Unmixed #2) and CRANAD-3 only (Unmixed #3).
Fig. 7 In vitro validation of CRET with ADLumin-1/CRANAD-3 pair. a Proposed CRET model between ADLumin-1 and CRANAD-3 with Aβ40 fibrils. Two nonconjugated molecules upon binding to fibrils to bring ADLumin-1 (Donor) close enough to CRANAD-3 (Acceptor) to initiate CRET; b Spectrum of the CRET pair with Aβ40 fibrils in PBS (red line), and the peak was consistent with the emission of CRANAD-3 in the presence of Aβ40 fibrils. Chemiluminescence spectrum of ADLumin-1 with Aβ40 fibrils (black line); and spectrum of the mixture of ADLumin-1 and CRANAD-3 without Aβ40 fibrils (pink line). The FRETing efficiency was very high, evident by the low intensity at 500–560 nm range; c Spectrum of the CRET pair in brain homogenate (red line), evident by a decrease in ADLumin-1 emission (black line) and increase in CRANAD-3 emission; d Spectral unmixing of DAS-CRET to separate the contribution from ADLumin-1 only (Unmixed #1), and CRET (Unmixed #2) and CRANAD-3 only (Unmixed #3).CC BY · PubMed Central
Fig. 8 Observation of CRET under in vivo mimic conditions. a A mixture of ADLumin-1 and CRANAD-3 with Aβ40 aggregates (CRET + Aβ) were injected subcutaneously into the right inner thigh of a female nude mouse and the control group of ADLumin-1+Aβ was injected into the left side; b The mouse was imaged from both the ventral and dorsal sides; c Spectral unmixing was conducted with sequence imaging at 15 min post injection to separate signals from ADLumin-1 (left, green) and from CRET pair (middle, red), and composite image of the two unmixed components (right); D Spectra of the unmixed contributors, ADLumin-1+Aβ (green), and CRET + Aβ (red).
Fig. 8 Observation of CRET under in vivo mimic conditions. a A mixture of ADLumin-1 and CRANAD-3 with Aβ40 aggregates (CRET + Aβ) were injected subcutaneously into the right inner thigh of a female nude mouse and the control group of ADLumin-1+Aβ was injected into the left side; b The mouse was imaged from both the ventral and dorsal sides; c Spectral unmixing was conducted with sequence imaging at 15 min post injection to separate signals from ADLumin-1 (left, green) and from CRET pair (middle, red), and composite image of the two unmixed components (right); D Spectra of the unmixed contributors, ADLumin-1+Aβ (green), and CRET + Aβ (red).CC BY · PubMed Central
Fig. 9 In vivo DAS-CRET imaging. a In vivo brain imaging of WT and 5XFAD mice with mixture of ADLumin-1 and CRANAD-3; b In vivo emission spectra of CRET pair (red line-5xFAD) and control (black line-WT), and ADLumin-1 only (pink line-5xFAD) and ADLumin-1 only control (blue line-WT); c–e Quantitative analysis of the images with CRET pair with the setting of open filter (c), at 640 nm (d) and after spectral unmixing (e). Data are represented as mean ± s.d. with n = 3 biologically independent animals. P = 0.0059 (c), P = 0.0292 (d), P = 0.0133 (e). The P-values were generated by Graphpad Prism 8 with two-tailed unpaired t-test; *P-value < 0.05, **P-value < 0.01. Source data underlying c–e are provided as Source Data file.
Fig. 9 In vivo DAS-CRET imaging. a In vivo brain imaging of WT and 5XFAD mice with mixture of ADLumin-1 and CRANAD-3; b In vivo emission spectra of CRET pair (red line-5xFAD) and control (black line-WT), and ADLumin-1 only (pink line-5xFAD) and ADLumin-1 only control (blue line-WT); c–e Quantitative analysis of the images with CRET pair with the setting of open filter (c), at 640 nm (d) and after spectral unmixing (e). Data are represented as mean ± s.d. with n = 3 biologically independent animals. P = 0.0059 (c), P = 0.0292 (d), P = 0.0133 (e). The P-values were generated by Graphpad Prism 8 with two-tailed unpaired t-test; *P-value < 0.05, **P-value < 0.01. Source data underlying c–e are provided as Source Data file.CC BY · PubMed Central

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Metadata

Authors
Yang, J., Yin, W., Van, R., Yin, K., Wang, P., Zheng, C., Zhu, B., Ran, K., Zhang, C., Kumar, M., Shao, Y., & Ran, C.
Affiliations
  • Harvard University (US)
  • University of Oklahoma (US)
  • Massachusetts Institute of Technology (US)
Corresponding author
Ran, Chongzhao
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2020-08-13
Volume
11
Issue
1
Pages
4052-4052
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-020-17783-4
PMC
PMC7426431
OpenAlex
W3048903908
Cited by
123
References
73
Citations by year
2026: 16 · 2025: 16 · 2024: 24 · 2023: 24 · 2022: 13 · 2021: 27 · 2020: 3
FWCI
10.07
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • ChemRxiv (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by-nc-nd)
Funders
U.S. Department of Health & Human Services | NIH | National Institute on Aging (R21AG059134, R01AG055413)
Topics
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Alzheimer's disease research and treatments
  • Physical Sciences › Chemistry › Spectroscopy › Molecular Sensors and Ion Detection
  • Physical Sciences › Materials Science › Materials Chemistry › Luminescence and Fluorescent Materials
Keywords
chemiluminescence probe, amyloid-beta detection, chemiluminescence resonance energy transfer, two-photon imaging, blood-brain barrier penetration, cerebral amyloid angiopathy, amyloid plaques, 5xFAD mice, in vivo brain imaging
MeSH terms
Animals, Luminescent Measurements, Amyloid beta-Peptides, Luminescence, Mice, Molecular Imaging, Optical Imaging, Protein Aggregates

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