Split prism-assisted optical aptasensor for ultrasensitive and reliable detection of SARS-CoV-2

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to substantial global mortality, emphasizing the need for rapid and sensitive detection. Reverse transcription–quantitative polymerase chain reaction (RT-qPCR), the current gold standard, is costly and time-consuming due to its complex procedures and resource requirements. In this study, a real-time split prism-assisted solution-immersed silicon (SP-SIS) aptasensor was developed for ultrasensitive and affordable detection of SARS-CoV-2 spike proteins. The newly designed split prism incorporates a thin metallic plate between optical components to reflect and absorb background noise originating from inner prism reflections. To enhance biosensing accuracy, the incident angle was precisely aligned with a low variation of ±0.005°, ensuring consistent optical performance across newly assembled sensors. Additionally, a surface with a controlled water contact angle significantly improved detection efficiency by promoting effective aptamer immobilization. The SP-SIS platform achieved detection limits of 76.8 fg/mL and 15.6 fg/mL for spike proteins, and 22.1 particles/mL and 28.7 particles/mL for virus-like particles, in viral transport medium and COVID-19-negative nasopharyngeal swab matrices, respectively. Moreover, it reliably detected SARS-CoV-2 variants and distinguished them from other coronaviruses, confirming high specificity. These findings demonstrate that the SP-SIS aptasensor provides a rapid, accurate, and ultrasensitive tool for early SARS-CoV-2 detection and can be extended to monitor emerging infectious diseases. • We developed a real-time SP-SIS aptasensor enabling ultrasensitive detection of SARS-CoV-2 spike proteins. • The split prism was designed with a thin metal plate between two optical components to eliminate background noise. • The incident angle was precisely aligned with a variation as low as ±0.005° for each newly attached sensor assembly. • We improved aptamer immobilization by tuning surface hydrodynamics, thereby enhancing detection efficiency. • The detection limits were 15.6 fg/mL for spike proteins and 28.7 particles/mL for virus-like particles.