Peer review of: Single-molecule photoreaction quantitation through intraparticle-surface energy transfer (i-SET) s  (2020)



RR2
 REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author):
Reviewer #2 (Remarks to the Author):
  The point-by-point response to the referees comments is satisfactory and so the revised
 manuscript is recommended for publication in Nature Communications.
  The article has been corected in response to reviewers comments. Despite I have some further
 questions, these questions do not change my opinion about high quality of this research and
 opinion this article is suitable for Nature Communication.
 1. Concerning my previous Q3 about Figure S20 - I understand that the models for direct FRET
 and i-FRET require differences, but still I want to know more, what this graph is teaching us. For
 example the direct FRET in YbEr with Y only shell (the YbER@Y NPs) will depend from Y shell
 thickness apart from NP diameter - so the presented 'picture' of phenomenon is not complete.
 Moreover, in Fig.S20b, the ET Efficiency decreases for rising size of NP because of increasing the
 surface area of a NP for a given amount of acceptor - kind of "diluting" the acceptors per NP
 surface (ET Eff/miu) and decreasing the relative contribution of Tb emission being quenched by
 acceptor to the total of all emitting Tb ions - is that what the model suggests? What is then the
 optimum balanced NP size, when one takes into account the brightness of NPs (which is rising for
 rising NP diameter, I guess) vs decreasing FRET efficiency. I think this is important to use a
 reliable model to predict/compare the properties of the system, but here more critical discussion is
 then needed.
 2. Concerning Fig.2f - Taking into account the that fact the multiple superficial donor ions (either
 Er or Tb) may transfer the energy to a single acceptor molecule (many-to-one or many-to-few),
 and the fact the lifetimes of both lanthanides are in 0.1-1 ms range, while that of acceptor is in the
 ~1 ns range, I would say, that in both cases, the acceptor brightness shall saturate only for larger
 A quantities, but not for single acceptor molecules, as many multiple-Ln-single-BDP FRET events
 have a chance to take place within the average lifetime of excited state on Ln. Approximately up to
 0.1-1ms/1ns ~= 10^5-10^6 times (per lifetime of excited state of Ln3+) the FRET may occur
 between D and A. Then, when acceptor receives the energy from donor, returns (within single ns)
 to ground state (emitting photons) and becomes ready for the next FRET event. Therefore, in my
 opinion, the brightness of acceptor should not (for low concentrations of acceptors) depend
 (decrease here - Fig.2f) on its concentration in these circumstances. Can the authors comment on
 this? Can you support your modelling with experimental data in this area?
 Concerning the suggestion by the reviewer #3 - Dexter energy (or Dexter electron) transfer is a
 fluorescence quenching, in which excited electron of the donor is transferred to acceptor via non-
 radiative way. Firstly, this happens typically within short 1 nm distance, secondly, optically active
 electrons of the donor (here Tb3+) are f-electrons, which do not participate in chemical reactions
 and are covered/protected by higher laying orbitals, thus I am not sure, that using DET to explain
 the ET here is correct. Tb3+ is one of rare earth ions that may change the oxidation state (to +4),
 indeed, but it seems it does not apply here.
 Reviewer #3 (Remarks to the Author):
 I like this paper and offered a generally positive review along with some specific suggestions
 where I thought the paper needed improvement. I’m surprised to see a couple of those
 suggestions were not addressed in the paper, as these do not involve new experiments. I think

 this paper should be published, after dealing with these specifics.
 For #2, the authors should add in a figure with difference spectra (ie, between specta with and
 without the fluorophores). I would like to see the isolated BODIPY peaks. Are the peaks where the
 BODIPY peaks are if the fluorophore is excited directly, or is there some other electronic effect that
 shifts the peak for the fluorophores on the nanoparticle surface? That was the key point.
 I’ve not seen a signal:noise ratio used for a cuvette measurement before, and I think it is odd to
 include it. S/N is useful in microscopy because highly localized signal (eg, an individual pixel) is
 often difficult to attribute to a specific source in the absence of integration across a large sample
 volume like a cuvette. If the authors require a S/N measurement even in a cuvette, this makes me
 less excited about the current results. I do not think it helps them to highlight this.
 For #5, my point was that recent work (the references I suggested) has shown that the concept
 “concentration quenching” is sometimes unimportant for core/shell UCNP structures. Yet the
 authors add these references to a sentence about concentration quenching, a concept those
 references specifically do not support.
 I would also double check the references, as I notice some of the names are incorrect.
 2) “As shown in Figure 2c, even at a low BDP concentration of ~0.5 BDP per nanoparticle, the
 sensitized
 emission from BDP molecules is still detectable with a signal-to-noise ratio of up to 30.”
 Signal:noise is not the correct term for the calculation in 2c. For cuvette measurements, the noise
 of a fully background-subtracted spectrum is 0, so signal:noise would be infinite. More appropriate
 here would be a difference spectrum to see if the peak matches the free BODIPY dye emission,
 and to calculate the ET efficiency. This BODIPY has an unfortunately small Stokes shift, which may
 make calculations of ET efficiency difficult.
 Response: We apologize for the misunderstanding caused by the unclear interpretation about the
 signalto-
 noise ratio. The discussion of sensitized emission of samples with low BDP loading concentration is
 aimed to indicate high detection limit of our system rather than calculate the ET efficiency (the
 study of
 ET efficiency was presented in Figure 2d). We agree with the reviewer that for cuvette
 measurements the
 background spectrum can be subtracted. However, even with a full subtraction the background
 noise is still not completely 0. There is a small fluctuation in the baseline spectrum due to the
 instrument response. For example, in a typical case of measurement we get a background
 fluctuation of about Å}20 counts (see Figure R5 below). Herein, in order to obtain a meaningful
 signal from the sensitized emission, the detectable emission intensity of dye molecules must be at
 least 3-10 times higher than the fluctuation of the background spectrum. In this experiment, we
 observed the sensitized emission of BDP is over 30 times higher than the background spectrum
 even when the loading concentration of BDP is as low as 0.5 per nanoparticle. We believe this is a
 good indication implying that our system can work for very low detection limit. Nevertheless, we
 have noted that the detection limit of the instrument is correlated to the molar concentration of
 dye molecule in solution. For clarity, we have amended the discussion in the manuscript as
 follows:
 “As shown in Figure 2c, the sensitized emission signal from the BDP molecules is around 30 times
 higher than the background spectrum (S/N = 30) even when the loading concentration is as low as
 ~0.5 BDP per nanoparticle (corresponding to ~500 nM BDP in solution). The result suggests the
 possibility of achieving highly sensitive FRET detection by adopting i-SET.”
 5) “Nevertheless, for conventional Er3+, Tm3+, or Ho3+ activators co-doped with Yb3+ or Nd3+

 ions as
 sensitizers21-23, a high content of activator and sensitizer combination would generally lead to
 low
 luminescence efficiencies of the nanoparticles due to concentration quenching effects24.”
 There is a growing literature at this point on high-Ln UCNPs, and it has become clear that
 concentration
 quenching is not a significant factor in core/shell UCNPs. The authors seem to recognize that
 adding Ybfree shells necessarily cuts off the prime energy loss pathway. See e.g., JACS 139, 3275
 (2017) and Nat Comm 9, 3082 (2018) for previous, in-depth discussions of high Ln core/shell
 UCNPs and why the concept of concentration quenching is dated. I think the discussion in the
 current paper can take this prior work into account to substantially shorten this part of the
 manuscript.
 Response: We thank the reviewer for the useful references. As suggested, we have cited these two
 papers
 as references 26 and 31 in the revised manuscript, and shorten the description about the
 concentration quenching in introduction as follows:
 “Nevertheless, for conventional Er3+, Tm3+, or Ho3+ activators co-doped with Yb3+ or Nd3+ ions
 as sensitizers22-24, a high content of activator and sensitizer combination would generally lead to
 low luminescence efficiencies of the nanoparticles due to concentration quenching effects25-31. It
 mainly conducts through coupled energy transfer from the lower intermediate states of
 sensitizers/activators to –OH and –CHn vibration modes of surface molecules/defects
 (Supplementary Scheme. S1 and 2).”