The current nucleic acid signal amplification methods for SARS-CoV-2 RNA detection heavily rely on the functions of biological enzymes which imposes stringent transportation and storage conditions, high cost and global supply shortages. Here, a non-enzymatic whole genome detection method based on a simple isothermal signal amplification approach is developed for rapid detection of SARS-CoV-2 RNA and potentially any types of nucleic acids regardless of their size. The assay, termed non-enzymatic isothermal strand displacement and amplification (NISDA), is able to quantify 10 RNA copies.µL⁻¹. In 164 clinical oropharyngeal RNA samples, NISDA assay is 100 % specific, and it is 96.77% and 100% sensitive when setting up in the laboratory and hospital, respectively. The NISDA assay does not require RNA reverse-transcription step and is fast (1 month), isothermal (42 °C) and user-friendly, making it an excellent assay for broad-based testing.

Controlling the selective one‐to‐one conjugation of RNA with nanoparticles is vital for future applications of RNA nanotechnology. Here, the monofunctionalization of a gold nanoparticle (AuNP) with a single copy of RNA is developed for ultrasensitive microRNA‐155 quantification using electrochemical surface‐enhanced Raman spectroscopy (EC‐SERS). A single AuNP is conjugated with one copy of the packaging RNA (pRNA) three‐way junction (RNA 3WJ). pRNA 3WJ containing one strand of the 3WJ is connected to a Sephadex G100 aptamer and a biotin group at each arm (SEPapt/3WJ/Bio) which is then immobilized to the Sephadex G100 resin. The resulting complex is connected to streptavidin‐coated AuNP (STV/AuNP). Next, the STV/AuNP–Bio/3WJa is purified and reassembled with another 3WJ to form a single‐labeled 3WJ/AuNP. Later, the monoconjugate is immobilized onto the AuNP‐electrodeposited indium tin oxide coated substrate for detecting microRNA‐155 based on EC‐SERS. Application of an optimum potential of +0.2 V results in extraordinary amplification (≈7 times) of methylene blue (reporter) SERS signal compared to the normal SERS signal. As a result, a highly sensitive detection of 60 × 10⁻¹⁸ m microRNA‐155 in 1 h in serum based on monoconjugated AuNP/RNA is achieved. Thus, the monofunctionalization of RNA onto nanoparticle can provide a new methodology for biosensor construction and diverse RNA nanotechnology development.