I'm surprised that you find ATP to be fluorescent. It is not fluorescent in an aqueous environment, so maybe it's fluorescence has to do with being in the carbon nanostructure. Or maybe it is the calixarene that is fluorescent.

Anyway, since the absorbance maximum of ATP is about 260 nM and calixarenes are aromatic compounds, I would suppose that at least part of the fluorescence quenching you are seeing is the inner filter effect, which is the absorption of excitation and/or emission light of the fluorescent compound by the other compound.

If it's the calixarene that is fluorescent in your experiment, rather than the ATP, you could still get fluorescence quenching of calixarene by itself due to the inner filter effect. Dynamic and static quenching are also possibilities.

Andrii Lesiuk

We have worked with calixarenes depending on the concentration. We studied their aggregation. The DLS method shows the aggregation of molecules up to sizes up to 500-1000 nm. The SAXS method does not show aggregation. The impression is that calixarene molecules with water form a gel with a nuclear quantum effect (probabilistic structure).

The fluorescence quenching of ATP by calix[4]arene-107 that you observed is likely due to a phenomenon called static quenching, which occurs when the quencher (in this case, the calixarene) binds to the fluorophore (ATP) in a non-radiative manner, thereby reducing the intensity of the fluorescence. The binding energy can be calculated from the quenching constant, which is related to the observed fluorescence quenching.

The specific shape of the dependence of I_0/I vs. calix concentration that you observed (Г-shape) can be explained by the concept of "quenching zones". The quenching zones are the regions on the concentration where the quencher concentration is high enough to bind to the fluorophore but not too high to cause a complete quenching. So it is possible that the quenching is not complete, and that the observed fluorescence is due to a balance between the binding and non-binding of the calixarene to the ATP.

To find the binding energy, you could use various methods such as Fluorescence Quenching Analysis (FQA), Fluorescence Lifetime Analysis (FLA), or Fluorescence Resonance Energy Transfer Analysis (FRET). Each of these methods provides different information on the binding dynamics, and the most appropriate method will depend on the specific system you are studying and the research question you are trying to answer.

You may also want to consider including control experiments to understand the nature of quenching, like measuring the fluorescence spectra of the quencher alone or quencher with different concentrations of the fluorophore. Additionally, you may want to consult the literature and experts in the field for guidance on appropriate methods and data analysis.

Adam B Shapiro , we detect emission spectra after irradiating the sample with 285 nm. Intensity maximum is at 395 nm and it decreases with increasing concentration of calixarenes.

From this, it seems that the fluorescence is coming from the calixarene rather than the ATP, which has an absorption maximum at about 260 nm, not 285 nm, unless ATP in the ATP-calixarene complex behaves differently from ATP in aqueous solution. Is calixarene without ATP fluorescent?

Adam B Shapiro , we measure emission at lambda_excitation=285 nm because at 260 nm we can't get luminescence of ATP. Firstly, with pure ATP, and then with addition of calixarene (+ DMSO because of bad solubility of C-107 in water) into the aqueous solutions. When concentration of calixarene increases the intensity drops.

As I said before, my experience is that ATP is not fluorescent in aqueous medium. If it were, a dilute solution would show a maximum excitation at 260 nm.

Adam B Shapiro , I have found at least this article about ATP fluorescence

Article Modification of the Intrinsic Fluorescence and the Biochemic...