Figure 1
Overview of the spectroscopic techniques. (a) Comparison of the assay dynamics in different standard matrices utilising the absorbance unit (AU) readings obtained from DEPC water, revised simulated body fluid (r-SBF), revised simulated body fluid adjusted with albumin (r-SBFA) and human plasma, all spiked with 50 μL of 376.8 nM β-NAD standard (S2). (b) Fluorescence scan of the autofluorescence of the Master Mix (MM) and a NAD+ sample as prepared in the actual assay reaction, scanned from λex1 = 280 nm − λex2 = 850 nm in steps of Δλ = 2 nm. The resorufin signal is indicated at λ = 590 nm in the detail view. (c) Enzyme dependent assay kinetics for β-NAD standards S1–S6 including a heparinised plasma sample and the blank of a given run using the fluorimetric method. (d) Enzyme dependent assay kinetics for β-NAD standards S1–S6 including a heparinised plasma sample and the blank of a given run using the colorimetric method. (e) Fluorescence scan of the autofluorescence of the Master Mix (MM) and β-NAD standard S1 as prepared in the actual assay reaction (S1 + r-SBFA + MM), scanned from λex1 = 280 nm − λex2 = 850 nm in steps of Δλ = 2 nm. (f) Visual representation of the resorufin quenching effect that occurs when a minuscule amount of heparinised plasma sample is added to the β-NAD standard S1.
