Label-Free Digital Detection of Intact Virions by Enhanced Scattering Microscopy

Abstract

Several applications in health diagnostics, food, safety, and environmental monitoring require rapid, simple, selective, and quantitatively accurate viral load monitoring. Here, we introduce the first label-free biosensing method that rapidly detects and quantifies intact virus in human saliva with single-virion resolution. Using pseudotype SARS-CoV-2 as a representative target, we immobilize aptamers with the ability to differentiate active from inactive virions on a photonic crystal, where the virions are captured through affinity with the spike protein displayed on the outer surface. Once captured, the intrinsic scattering of the virions is amplified and detected through interferometric imaging. Our approach analyzes the motion trajectory of each captured virion, enabling highly selective recognition against nontarget virions, while providing a limit of detection of 1 × 10 3 copies/mL at room temperature. The approach offers an alternative to enzymatic amplification assays for point-of-collection diagnostics.

Aptamers targeting SARS-COV-2: a promising tool to fight against COVID-19

“…A SPR aptasensor has also been constructed for the detection of N protein of SARS-CoV-2 by using niobium carbide MXene quantum dots to anchor aptamer A58 (Table 2), resulting in a LOD of 4.9 pg/ml for N protein [46]. Another label-free biosensing method was developed, called photonic resonator interferometric scattering microscopy (PRISM), that uses the aptamer SARS2-AR10 (Table 2) and a photonic crystal as the transducer substrate, and allowed real-time visualization and mass quantification of SARS-CoV-2 virions (LOD 1 × 10 3 copies/ml) [47]. Although the aforementioned SERS-based, SPR-based, or PRISM-based aptasensors provide promising detection results compared with LFA and ELISA, detecting the optical signals requires advanced, specialized, and costly equipment.…”

Aptamers for SARS‐CoV‐2: Isolation, Characterization, and Diagnostic and Therapeutic Developments

“…A recent assay based on aptamer‐coated photonic crystals has been used for interferometric imaging of the scattering of bound pseudotyped SARS‐CoV‐2, showing single virion resolution and a detection limit of 10 3 copies of virions per mL with no need for amplification [120] . The assay did not require labelling of any species, but was relatively slow and labour intensive, making it difficult to implement for rapid, large‐scale testing of infections.…”

Microscopies Enabled by Photonic Metamaterials

“…Cunningham and colleagues demonstrated observation of single AuNPs, proteins [71], and virions [74] using PRISM. AuNPs with diameters down to 5 nm can be individually discerned from the background noise.…”