An ultrasensitive fluorescence aptasensor for SARS-CoV-2 antigen based on hyperbranched rolling circle amplification

Area of Science:

• Biomedical Engineering
• Molecular Diagnostics
• Infectious Disease Research

Background:

• Accurate early diagnosis of SARS-CoV-2 (COVID-19) is crucial for mitigating disease impact.
• Direct viral antigen detection offers a more accurate reflection of infectivity compared to RNA or antibody tests.
• Developing sensitive and convenient biosensors for SARS-CoV-2 antigens remains a significant challenge.

Purpose of the Study:

• To develop a simple, ultrasensitive, and quantitative aptasensor for detecting SARS-CoV-2 S1 protein and pseudovirus.
• To establish a novel diagnostic strategy for early-stage SARS-CoV-2 infection.
• To demonstrate the potential of aptasensors for detecting other viral targets.

Main Methods:

• Selection of an optimal aptamer sequence using enzyme-linked oligonucleotide assay and molecular docking simulation.
• Development of a Human Rectal Cancer Associated antigen (HRCA)-based aptasensor utilizing a sandwich complex formation on microplates.
• Fluorescent detection of the target analytes via HRCA amplification.

Main Results:

• The aptasensor achieved an ultrasensitive limit of detection (LOD) of 89.7 fg/mL for S1 protein within a linear range of 100 fg/mL to 1 μg/mL.
• The sensor demonstrated a LOD of 51 thousand infectious units (TU)/μL for SARS-CoV-2 spike pseudovirus in artificial saliva.
• The developed aptasensor requires no complex operations or specialized equipment.

Conclusions:

• The HRCA-based aptasensor provides a simple, ultrasensitive, and quantitative method for detecting SARS-CoV-2 S1 protein and pseudovirus.
• This aptasensor holds significant potential for early-stage SARS-CoV-2 infection diagnosis, improving timeliness and accuracy.
• The study presents a viable strategy for developing aptasensors for various diagnostic targets.