Singlet oxygen-based photoelectrochemical biosensor for dengue virus RNA

Highlights
• Singlet oxygen-based photoelectrochemical biosensor for direct dengue RNA detection.
• Optimizing probe design is essential for overcoming secondary structures.
• The biosensor has an LOD of 7 pM, without requiring enzymes.
• High specificity against Zika virus.

Abstract
Rapid and accurate detection of dengue virus is essential for effective patient management and preventing outbreaks from escalating into epidemics or pandemics. However, RNA's instability and tendency to form stable secondary structures pose significant challenges in biosensor development. In this work, we present an enzyme-free, singlet oxygen-based photoelectrochemical (PEC) biosensor for direct RNA detection, eliminating the need for cDNA conversion, thermal denaturation, or enzymes. A key innovation lies in the probe design strategy, which acts on regions that promote unfolding of the target RNA, enhancing its accessibility and therefore the sensitivity of the sensor. We found that elongating a single detection probe in combination with a strategic binding site significantly improved PEC response rather than using a dual-detection probe design. A comparative study between an RNA and DNA target further revealed that backbone composition substantially impacts hybridization dynamics and PEC signal generation. The optimized biosensor achieved a limit of detection of 7 pM (4.2 × 106 copies/μL) for dengue virus serotype 1 RNA and demonstrated 96.8 % ± 0.4 % discrimination against Zika RNA at room temperature. We also identified critical assay components, such as the magnetic beads that contribute to background signals. This work advances PEC biosensing by establishing a robust, amplification-free platform for the detection of structured RNAs and introduces a generalizable strategy for probe design based on RNA structural accessibility.