Single Functionalized pRNA/Gold Nanoparticle for Ultrasensitive MicroRNA Detection Using Electrochemical Surface-Enhanced Raman Spectroscopy.

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 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.