Development of Nanoparticles Based Assays for the Direct Detection of Unamplified Nucleic Acids in Clinical Specimens

Abstract

Advances in molecular diagnostic technologies have led to substantial progress in the understanding and detection of a multitude of diseases including infectious, genetic diseases and cancer. These technologies have become a cornerstone in modern clinical diagnostics. These molecular detection strategies have the advantage of being sensitive and specific; however, they are all relatively costly, labor-intensive, and require a sophisticated laboratory infrastructure.
Nanotechnology is a powerful and rapidly evolving field whose integration into the molecular diagnostics arena would have a positive impact on the development of simpler effective nucleic acid testing. The utilization of nanoparticles would also allow assay miniaturization development of “at point of care” assays. One of the most promising nano-structures are gold nanoparticles (AuNPs) which exhibit unique optical properties. Moreover, magnetic nanoparticles (MNP), is demonstrating high potential for utility in molecular assays. They are characterized by Superparamagnetic properties, and is currently used for the selective and efficient capture of the target bio-molecule. Here we have developed two prototype nanoparticles based assays for direct detection of unamplified nucleic acids in clinical samples. The first was the direct detection of unamplified Hepatitis C Virus (HCV) RNA. This was done using two approaches; the first employed negatively charged unmodified AuNPs. The developed prototype has a sensitivity and specificity of 92% and 89.9% respectively, and a detection limit of 50 copies/reaction. The other developed prototype is a combination of MNPs for specific HCV RNA capture, and cationic AuNPs for detection. The assay prototype has a sensitivity and specificity of 96.5% and 96% respectively, with a detection limit of 15IU/ml blood (1 IU corresponds to 2-5 copies). Both approaches were tested on clinical serum samples from HCV infected patients. The second application was the detection of cancer biomarkers. We combined MNPs specific capture with unmodified negatively charged AuNPs for detection of two tumor biomarkers. The first was, Hepatoma Up Regulated Protein (HURP) RNA, that has been detected in clinical urine samples from urinary bladder cancer patients, with a sensitivity and specificity of 90% & 94% respectively. The other target was Histidine-Rich Glycoprotein (HRG) RNA, a breast cancer biomarker which was detected in breast tissues, with a sensitivity and specificity of 90%. The developed prototype is capable of differentiating between the various cancer stages, grades, and subtypes.