NADH biofluoro-shifting to red light toward multi-wavelength imaging application of VOCs

In breath and transdermal gas, which contain thousands of volatile organic compounds (VOCs), selective simultaneous measurement of multiple VOCs is considered effective for noninvasive pharmacokinetic and metabolic tracking. Enzymatic optical biosensors with high selectivity and sensitivity have potential for simultaneous sensing and imaging of multiple VOCs by wavelength discrimination, but most enzymatic optical biosensors emit blue light region (400–500 nm). In this study, we investigated the possibility of red shifting the wavelength of luminol chemiluminescence (CL) and NADH fluorescence (FL), which emits blue light, for multiplexed VOCs imaging. Luminol CL and NADH FL were converted to red by addition of rhodamine B and by resorufin (excitation 560 nm, fluorescence 590 nm) which induced by diaphorase (DP) with resazurin. The results showed that resorufin was suitable for multiplexing because the spectrum overlap with blue region was minimal. In addition, a DP-immobilized cotton mesh enabled spatiotemporal imaging of NADH mist spray at optimal of various conditions (buffer pH = 6.5, DP amount = 60 U/cm2, initial resazurin = 100 μM) with fast response (90% response time = 10 s). Furthermore, the NADH detection sensitivity was sufficient for VOCs imaging with red light in combination with NADH-dependent enzymes. In the future, this technique can be used for simultaneous imaging of multiple VOCs in the same region of interest.