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1¡¢The detection limits of both assays were determined by the 3s-method and found to be 10 fM. However, it is not stated if this analysis was done using (ligase) buffer or a real biological sample matrix like e.g. cell culture medium (that might / should be a ¡°real world matrix¡±) for sample dilution. As described on page 3 (5), line 25 and shown in Fig. 3B, cell culture fluid was (had to be) diluted 1:5 with ligase buffer, corrupting the found limits of detection by, at least, a factor of 5 for real world samples.
2¡¢Determination of the limit of detection (LOD) but also the lower limit of quantification (LLOQ)! should be performed using standards of ATP prepared in full cell culture fluid, only subsequently diluted in ligase buffer for final analysis.
3¡¢Furthermore, since the LODs found by the 3s-method might be based on a ¡°lucky¡± experiments using buffer?!-blanks only, dilution series should be run down to stated concentrations to demonstrate real significant differences between blank values (based on cell culture fluid) and the stated LOD (cp. Figure S8).
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