Exponential Amplification Using Photoredox Autocatalysis

Abstract

Exponential molecular amplification such as the polymerase chain reaction is a powerful tool that allows ultrasensitive biodetection. Here we report a new exponential amplification strategy based on photoredox autocatalysis, where eosin Y, a photocatalyst, amplifies itself by activating a non-fluorescent eosin Y derivative (EYH2) under green light. The deactivated photocatalyst is stable and rapidly activated under low intensity light, making the eosin Y amplification suitable for resource-limited settings. Through steady-state kinetic studies and reaction modeling, we found that EYH2 is either oxidized to eosin Y via one-electron oxidation by triplet eosin Y and subsequent 1e ─ /H + transfer, or activated by singlet oxygen with the risk of degradation. By reducing the rate of the EYH2 degradation, we successfully improved EYH2to-eosin Y recovery, achieving efficient autocatalytic eosin Y amplification. Additionally, to demonstrate its flexibility in output signals, we coupled the eosin Y amplification with photo-induced chromogenic polymerization, enabling sensitive visual detection of analytes. Finally, we applied the exponential amplification methods in developing bioassays for detection of biomarkers including SARS-CoV-2 nucleocapsid protein, an antigen used in the diagnosis of COVID-19. ASSOCIATED CONTENTSupporting Information. Materials and Methods; Model development; Table S1; Figures S1-S24. This material is available free of charge via the Internet at http://pubs.acs.org.

Dual Photoredox Catalysis Strategy for Enhanced Photopolymerization-Based Colorimetric Biodetection

“…Furthermore, by making the abovementioned solutions in a D 2 O-based buffer, we increased the lifetime of singlet oxygen produced by energy transfer from triplet MB + to ground-state oxygen. From this experiment, we confirmed that the concentrated TEOA (150 mM) compared to dissolved oxygen (0.25 mM) in both H 2 O-based and D 2 O-based buffer solutions − could make the EY amplification less vulnerable to the singlet oxygen-driven degradation of EY 2– and EYH 3– (Figure B) because a larger portion of triplet MB + would be quenched by TEOA, not by oxygen, retarding the generation of singlet oxygen.…”

“…These proof-of-concept assays reveal that the LoD of PBA can be successfully improved by replacing submicromolar EY 2– with 12 μM EYH 3– and amplifying EY 2– concentration in situ via the MB + photocatalysis. The MB + -specific EY amplification demonstrated here not only enables 100- to 500-fold enhancement in sensitivity but also provides high practicality compared to liposome-enhanced PBA in that the EYH 3– reagent can be stored at room temperature in contrast to liposomes that should be stored at 4 °C. Furthermore, the amplification time for the new method is still reasonably short (5 min under red light + 40 s under green light for the paper-based tests), and low-cost portable light sources can be developed for the dual photoredox catalysis.…”

“…EYH 3– was synthesized using the method described previously . An oven-dried round-bottom flask containing a stir bar was charged with sodium borohydride (4.54 g, 0.12 mol) and 1 mM eosin Y (EY 2– ) solution (120 mL, pH 11).…”

“…Alternative approaches for improving the LoD of PBA would be to remove the additional EY 2– and instead amplify the photocatalyst in situ in the presence of specifically captured target molecules to overcome the oxygen inhibition. One way to incorporate this photocatalyst amplification strategy into PBA is to employ photoredox autocatalysis, where a non-fluorescent derivative (dihydroeosin Y, EYH 3– ) of EY 2– is autocatalytically oxidized to EY 2– by target-associated EY 2– under green light . Given the high sensitivity of this exponential amplification, however, this approach could potentially be limited by high background signals generated from small impurity (0.05%) of EY 2– in the EYH 3– stock …”

“…One way to incorporate this photocatalyst amplification strategy into PBA is to employ photoredox autocatalysis, where a non-fluorescent derivative (dihydroeosin Y, EYH 3– ) of EY 2– is autocatalytically oxidized to EY 2– by target-associated EY 2– under green light . Given the high sensitivity of this exponential amplification, however, this approach could potentially be limited by high background signals generated from small impurity (0.05%) of EY 2– in the EYH 3– stock …”