oa Comparing two colorimetric AuNPs-based assays for direct detection of unamplified hepatitis C virus RNA in clinical specimens

Background. Two different approaches for direct detection of unamplified hepatitis C virus (HCV) RNA have been developed using two types of spherical gold nanoparticles (AuNPs); negatively charged AuNPs [1] and cationic AuNPs [2]. Both assays have demonstrated good clinical performance, cost-effectiveness and short turnaround time compared to routine amplification-based molecular assays. AuNPs-based assays utilize the phenomenon of surface plasmon resonance (SPR) which is the origin of the unique optical properties of AuNPs and responsible for their color change from red to blue upon aggregation. However, the assay's target detection mechanism differs according to the surface charge of AuNPs. Methods. In this work, a total of 22 serum samples (11 HCV-positive, 11 HCV-negative) were characterized using real-time PCR and tested using both AuNPs-based approaches to compare and evaluate their performance in direct colorimetric HCV RNA detection. Negatively charged spherical AuNPs were synthesized using the citrate reduction method [3], and cationic spherical AuNPs were synthesized as described previously [4]. The prepared AuNPs were characterized using UV spectrophotometry and scanning electron microscopy. Total RNA was extracted from the serum samples, and real-time HCV PCR was performed. The extracted RNA was then assayed using the anionic AuNPs. For the cationic AuNPs approach, a modified extraction protocol that employed magnetic nanoparticles and a specificity-conferring HCV-specific oligonucleotide was used prior to addition of the cationic AuNPs. Results. The turnaround time using anionic AuNPs was about an hour, while that using cationic AuNPs was about 45 minutes. The anionic AuNP assay detected 90.9% and 81.8% of positive and negative samples; respectively. The cationic AuNP assay detected 90.9% and 100% of positive and negative HCV samples, respectively. Conclusion. The cationic AuNP assay showed better clinical performance than anionic AuNP assay for detection of unamplified HCV RNA in serum, particularly to rule out viral infection, which warrants further investigation. References: References 1. Shawky SM, Bald D, Azzazy HME. Direct detection of unamplified hepatitis C virus RNA using unmodified gold nanoparticles. Clin Biochem 2010;43: 1163-8. 2. Shawky SM, Guirgis BSS, Azzazy HME. Specific detection of unamplified HCV RNA using colloidal cationic gold nanoparticles. 19th International Symposium on Hepatitis C Virus and Related Viruses. Venice, Italy, 2012. 3. Storhoff JJ, Elghanian R, Mucic RC, Mirkin CA, Letsinger RL. One-pot colorimetric differentiation of polynucleotides with single base imperfections using gold nanoparticle probes. J Am Chem Soc 1998;120:1959-64. 4. Huang X JP, El-Sayed IH, El-Sayed MA. Gold nanoparticles: interesting optical properties and recent applications in cancer diagnostics and therapy. Nanomed 2007;2.