Peer review of: Turn-on chemiluminescence probes and dual-amplification of signal for detection of amyloid beta (2020)

REVIEWER COMMENTS 

Reviewer #1 (Remarks to the Author): 

In this work, the authors reported about the design and development of a new chemiluminescent 
probe (ADLumin-1) that is able to “turn on” its chemiluminescence in presence of the target 
analyte (A\beta) and they tested this approach both in vitro and in vivo. Moreover, to overcome 
the limitation of short emission of ADLumin-1, they demonstrated the feasibility to achieve dual-
amplification of signal via chemiluminescence resonance energy transfer (DAS-CRET) with two 
non-conjugated smart probes in solutions, tissues and brain homogenates, and in vivo whole brain 
imaging. 

This work can be of great interest for different sectors: for imaging-based diagnostics but also for 
the synthesis of a new generation of chemiluminescent tracers that activate themselves in the 
presence of the target analyte, making them independent of the addition of other reagents, paving 
the way for a reagentless approach. This may represent a turning point in the field of the use of 
chemiluminescence as an ideal optical detection method which thus becomes highly competitive 
with colorimetric or fluorescent based methods. Indeed, although ADLumin-1 was designed for 
A\beta s, the authors believe that this strategy could be potentially extended to a wide range of 
targets. 

The discussion is very thorough, the statistics of the mice used for the experiments is not very 
extensive but can be used as proof of concept. It is a very large work, which includes the synthesis 
of the chemiluminescent marker, its photophysical characterization and in vitro and in vivo tests. 
The steps are well detailed and exposed in a logical and orderly way. 

The only thing that in my opinion deserves a deepening is the topic of in vivo toxicity of these 
molecules. Biocompatibility is indeed a crucial aspect in the design and development of markers to 
be used in vivo. 


Reviewer #2 (Remarks to the Author): 

This paper describes the evaluation of two (mostly one) new chemiluminescent compounds that 
putatively bind to Abeta species and exhibit enhanced emission of light when bound compared to 
free in solution. The general idea is novel and the results represent a nice demonstration of the 
power of this approach for labeling of amyloids for optical detection in vitro and in vivo. While the 
results presented are fairly convincing, the manuscript could be improved significantly by inclusion 
of more thorough characterization of the compound-amyloid complex, increased discussion 
regarding the mechanism for enhanced luminescence, and general improvements in clarity of 
experiments and data presented. 

Overall, it is a very nice story and should be publishable after some careful revisions. 

The following are a few specific comments that the authors should consider: 

-Should provide direct evidence of binding to Abeta species. While the structure looks like it should 
bind to fibrils, the authors should provide some data to support what types of aggregates the 
ADLumin-X compounds bind. 

-Should provide some rationale (ideally with supporting data) for why the ADLumin-X compounds 
show enhanced chemiluminescence when bound to Abeta versus free in solution. Oxygen levels 
are the same whether the Abeta samples are present or not, so how could the presumed binding 
of the compounds to Abeta lead to enhanced chemiluminescence in the presence of O2? 
-Figure 1A, Moiety B implies that ADLumin-X intercalates between peptides within a larger beta 
sheet superstructure. This binding mode appears different than the computed binding mode shown 
in Figure 3C? The authors should reconcile this discrepancy and remain consistent throughout the 
manuscript. Same issue with Figure 7A. Is the binding mode for CRANAD-3 known? 

-Figure 5B: It is unclear what is being imaged here. Is this an image from a living 5xFAD mouse, 
as the figure caption and main text might suggest? What is the scale bar and how was this image 
acquired? How were the blood vessels stained? What region of the brain is being shown? 

-Figure 5C: If this image is an ex vivo slice of the mouse brain used to generate the image in 
Figure 5B, why aren’t the blood vessels shown differentially stained? What happened to the 
vascular plaques associated with CAA? 




Reviewer #3 (Remarks to the Author): 

In this manuscript, the authors reported a smart chemiluminescence probe for Abs in vitro and in 
vivo, in order to realize the NIR imaging, DAS-CRET was carried out for the detection of Abs (in 
the brain and eyes) in living 5XFAD mice. It’s certainly an interesting paper, but the following 
questions should be further clarified before acceptance in Nature Communications or other 
journals. 
1. ADLumin-1 can bind to Abs, but with lower affinity (Kd = 2.1 uM) compared with other Ab 
probes. In addition, according to the author's description and experimental results, ADLumin-1 
was very sensitive to oxygen, and quickly (within seconds) converted to ADLumin-3, and how is its 
affinity to Abs? 
2. In figure3B, why the chemiluminescence spectra of ADLumin-1 alone is very low? After reacting 
with DMSO, there should be some light released, and in addition, the chemiluminescence was 
dramatically turned-on with Aβ40 aggregates, that’s hard to understand, without the excitation 
light source, the energy is conserved, and the signal intensity should only depend on the 
concentration of ADLumin-1 and oxygen. 
3. Compared with figure3D and F, what is the major difference of oxygen or DMSO between PBS 
and mouse brain homogenate? 
4. In vivo chemiluminescence imaging with ADLumin-1 in figure 6A and figure 9A, in addition to 
the signal from the nose, the palm signal of 5XFAD mice seems to have a higher intensity than the 
WT group, quantitative data of these areas should be carefully analyzed to exclude the influence of 
administration dose. 
5. In figure9A, the eye signal of WT mice seems to have a higher intensity than 5XFAD, which is 
contrary to the result in figiure6A.