## 3 Results
### The Oxidation of Trp by Triplet State of Rib Probed by ns-TA Spectroscopy  
The irradiation of Rib solutions at 445 nm excites Rib to its lowest singlet excited state, which undergoes efficient intersystem crossing to produce the highly reactive \({}^{3}\)Rib\({}^{\bullet}\) species. This triplet state is critical in various photobiological processes [21,22]. The following oxidation processes of tryptophan by this triplet state of Rib were monitored using nanosecond time-resolved absorption spectroscopy. As illustrated in Figure 2, panels (A) and (B) present the complete transient absorption spectra for Rib and Rib upon Trp addition, respectively, displayed at various delay times. Figure 2A shows the transient absorption spectrum of the \({}^{3}\)Rib\({}^{\bullet}\) in PBS solution at pH 7.2. The 400-500 nm wavelength range feature corresponds to the ground-state bleaching signal of Rib, with a negative absorption peak value at 450 nm. The broader absorption band from 500 to 800 nm is attributed to the triplet state of the Rib, reaching maximum absorption at -720 nm [19]. In the presence of oxygen, the \({}^{3}\)Rib\({}^{\bullet}\) decays completely within 12 us, as displayed in the insert panel of Figure 2A, which is similar to the transient spectra reported for Rib in the literature [19]. In the addition of Trp, this approach to the reactive triplet state of \({}^{3}\)Rib\({}^{\bullet}\) enables real-time monitoring of its reaction with Trp via transient absorption spectroscopy (Figure 2B). The triplet lifetime of the Rib at 720 nm dropped to 2.7 us with a mono-exponential fitting to the decay curve (see Figure 2C). Compared to Figure 2A, a smaller absorption intensity of the \({}^{3}\)Rib\({}^{\bullet}\) can be observed in Figure 2B with an equivalent excitation condition, indicating that Trp exhibits a quenching effect on the \({}^{3}\)Rib\({}^{\bullet}\). In the absence of Trp, \({}^{3}\)Rib\({}^{\bullet}\) fully decays within 12 us (Figure 2A). However, upon the addition of Trp, a prominent long-lived excited state emerges at 12 us within the 500-600 nm range, as shown in the inset panel of Figure 2B, and remains up to 150 us. This state is attributed to the long lifetime of Trp radical cation (Trp\({}^{\bullet+}\)) [10, 11, 12]. The corresponding kinetics at characteristic wavelengths are illustrated in Figure 2D. Specifically, the kinetic profile at 460 nm indicates that the ground-state bleaching of Rib is not fully restored at a time scale of 20 us, which is likely due to the formation of RibH\({}^{\bullet}\) with transient features at -320 and 520 nm through a rapid protonation process [9]. The distinct feature of the Trp\({}^{\bullet+}\) at 560 nm was further confirmed by the sulfate radical anion with a significantly stronger oxidant (\(E^{0}\) = 2.43 V vs. NHE) [23], displaying a similar spectral feature and kinetics (Figure S1). Notably, we chose a relatively low Rib concentration of 1.0 \(\times\) 10\({}^{-5}\) M in this study; when the concentration of Trp is below 1.0 \(\times\) 10\({}^{-4}\) M, Rib does not effectively sensitize the formation of long-lived  
Figure 1: (**A**) Absorption spectra of riboflavin (Rib, 1.0 \(\times\) 10\({}^{-5}\) M), tryptophan (Trp, 1.0 \(\times\) 10\({}^{-4}\) M), syringic acid (Syr, 2.0 \(\times\) 10\({}^{-4}\) M), and vanillic acid (Van, 2.0 \(\times\) 10\({}^{-4}\) M). (**B**) The chemical formulae of Rib, Trp, Van, and Syr. All the samples were in PBS (pH = 7.2) buffer measured at room temperature. The excitation wavelength used in the transient spectrum is 445 nm, to selectively excite Rib without affecting other substances. Trp\({}^{\bullet+}\) (Figure S2). The decay of triplet Rib at 720 nm followed first-order exponential kinetics, both in the absence and presence of Trp, indicating that\({}^{3}\)Rib\({}^{\bullet}\) can transfer electrons to Trp. Consistently, the presence of certain amino acids accelerated the decay of \({}^{3}\)Rib\({}^{\bullet}\), with the decay rate increasing proportionally to the concentration of these amino acids [24].