Speaker Details

Biography

Dr. Azzazy is a full, tenured professor of Chemistry at the American University in Cairo (AUC). He is also the leader of the Novel Diagnostics & Therapeutics Research group at Yousef Jameel Science & Technology Research Center and the director of the International Medical Technologists training program. Dr. Azzazy is also an adjunct professor at the Graduate School of Information Technology and Management, University of Maryland University College, USA. He served as director of biotechnology discipline at the Medical & Research Technology department (University of Maryland), the chairman of Chemistry department, the founding director of M.Sc. Chemistry program, and the Associate Dean for Graduate Studies & Research at AUC. He has published 42 referred journal articles, 50 conference abstracts, 22 text book chapters, and 8 monographs. Dr. Azzazy is a member of the editorial board of the Clinical Biochemistry journal (Canada), Clin Chimica Acta (USA), and Clinical Chemistry Clinical Laboratory Medicine journal (Europe). Dr. Azzazy has received several awards including Upjohn Research Achievement Award (UNT), Graduate Faculty Award for the Outstanding Graduate (UNT-HSC), Clin Chem Chair’s Award (Board of Registry, American Society for Clinical Pathology), Excellence in Research and Creative Endeavors Award (AUC), and Excellence in Teaching Award (AUC). In 2011, he received the State Prize in Advanced Technological Sciences from the National Academy for Scientific Research & Technology. Dr. Azzazy received his B.Sc. and graduate diploma in Biochemistry from Alexandria University, Egypt. In 1994, he received his PhD in Biochemistry and Molecular Biology from University of North Texas Health Science Center (UNT-HSC) in Fort Worth, TX, USA. He is certified as a diplomate of the American Board of Clinical Chemistry, Washington, DC, USA in two specializations: clinical chemistry and molecular diagnostics. He is also certified as a Specialist in Clinical Chemistry (SC), Board of Registry, the American Society for Clinical Pathology (ASCP), Chicago, IL, USA. Dr. Azzazy is a fellow of the National Academy of Clinical Biochemistry (FACB), Washington, DC, USA

Abstract

Gold Nanoparticles: A Versatile Platform for Sensitive Biosensing

Gold nanoparticles (AuNPs) have unique optophysical properties, can be easily prepared, and different techniques are available for their detection. AuNPs can be chemically modified and functionalized for detection of biomolecules. Spherical AuNPs were synthesized by citrate reduction of HAuCl4 and characterized by SEM and spectrophotometry. Unmodified AuNPs were used for direct detection of nucleic acid targets or conjugated to antibodies for detection of protein targets. We have developed a rapid, simple and inexpensive assay for direct detection of unamplified hepatitis C virus (HCV) RNA extracted from clinical samples using unmodified AuNPs. Serum samples were collected from healthy volunteers (n=45) and chronic HCV patients (n=80). Extracted RNA and an oligotargeter targeting the 5'UTR of HCV were denatured and annealed then AuNPs added. In HCV positive specimens, the color of the solution changed from red to blue. The assay has a sensitivity of 92%, a specificity of 89%, and a detection limit of 50 copies/reaction. The developed assay is highly sensitive, has a turnaround time of <1 h, and eliminates the need for thermal cycling and detection instruments. Based on the ability of AuNPs to efficiently quench fluorescence, we have also developed a fluorimetric assay prototype for direct detection of HCV RNA in serum. Extracted HCV RNA was added to a hybridization buffer containing a FAM-labeled probe targeting a conserved sequence in the HCV 5’UTR. After denaturation, annealing, and cooling; AuNPs were added to the mixture and the fluorescence emission at 520 nm was measured. In HCV positive specimens, a strong fluorescence signal was detected as the probe hybridized to the target and the fluorescence was not quenched by the AuNPs. In HCV negative specimens, the probe remained adsorbed on AuNPs and its fluorescence was quenched. For detection of tuberculosis (TB), one of the most significant causes of morbidity and mortality worldwide, we have developed two assay prototypes for detection of TB DNA. Unmodified spherical AuNPs were used for direct and quick detection of Mycobacterium tuberculosis complex (MTBC) DNA after PCR amplification. A second assay detected TB DNA directly after restriction digestion of TB genomic DNA isolated from TB cultures using a single oligotargeter that recognizes 16s rDNA gene segment. Detection limits were 1 ng for PCR product and 40 ng for digested genomic DNA. The assay showed 100% sensitivity and 100% specificity as compared with bacterial culture method and semi-nested PCR. The developed assay prototype is simple, sensitive, rapid, and shows a great potential in the clinical diagnosis of TB especially in developing countries with low resource settings. A simple and sensitive immunoassay that successfully detects malaria antigens in infected blood cultures was developed. This homogeneous assay was based on quenching of cyanine 3B-labeled recombinant Plasmodium falciparum heat shock protein 70 upon binding to AuNPs functionalized with an anti-Hsp70 antibody. The competitive fluorescence immunoassay was successfully used for the detection of antigen in malaria-infected human blood cultures at a 3% parasitemia level, and is assumed to detect parasitemia levels as low as 0.23%. For detection of C-reactive protein (CRP), a marker of cardiovascular diseases, pure antigen in borate buffer or serum was added to AuNPs conjugated to anti-CRP antibodies. The AuNP-anti-CRP conjugates aggregated leading to an increase in the value of the absorbance ratio of aggregated AuNPs (600 nm) to non-aggregated AuNPs (532 nm). The assay was linear from 0-10 g/mL for CRP in buffer and 0-2 g/mL and 3-5 g/mL for CRP in serum. The assay is precise at low concentrations of CRP, simple to perform, and can be automated. In conclusion, AuNPs have shown great potential for developing sensitive, inexpensive diagnostic tests for fast detection of infectious agents and disease biomarkers. We have successfully developed promising nanogold assay prototypes for detection of HCV, tuberculosis, malaria, and CRP. AuNPs should be further explored for detection of different infectious agents and disease biomarkers.