Direct detection of unamplified hepatoma upregulated protein RNA in urine using gold nanoparticles for bladder cancer diagnosis Sanaa Eissa a , Sherif M. Shawky b , Marwa Matboli a , Shaymaa Mohamed a , Hassan M.E. Azzazy b,c, ⁎ a Oncology Diagnostic Unit, Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Ain Shams University, Abbassia, Cairo 11381, Egypt b Youssef Jameel Science & Technology Research Center, The American University in Cairo, P.O. Box 74, New Cairo 11835, Egypt c Department of Chemistry, The American University in Cairo, P.O. Box 74, New Cairo 11835, Egypt abstract article info Article history: Received 31 August 2013 Received in revised form 16 October 2013 Accepted 21 October 2013 Available online 29 October 2013 Keywords: Bladder carcinoma Gold nanoparticles Hepatoma upregulated protein Magnetic nanoparticles Squamous cell carcinoma Transitional cell carcinoma Urinary biomarkers Objective: To develop a gold nanoparticle (AuNP) assay for direct detection of unamplified HURP RNA in urine. Design and methods: HURP RNA was extracted from urine samples (50 bladder carcinoma patients, 25 be- nign bladder lesions, and 25 controls) and further purified using magnetic nanoparticles (MNPs), functionalized with HURP RNA-specific oligonucleotides, and then detected by RT-PCR or gold nanoparticles. Results: The developed HURP RNA AuNP assay has a sensitivity and a specificity of 88.5% and 94%, respectively, and a detection limit of 2.4 nmol/L. The concordance between the HURP AuNP assay with RT-PCR after RNA purification using functionalized MNPs was 97%. Conclusions: The developed colorimetric HURP RNA AuNP assay is sensitive, simple, and can aid noninvasive diagnosis of bladder cancer. © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Introduction Bladder carcinoma is among the five most common malignancies worldwide. It is the second most common tumor of the genitourinary tract and the second most common cause of death in patients with gen- itourinary tract malignancies. Classical cytology and cystoscopy are mainly used for the surveil- lance of patients with bladder cancer. The low sensitivity for low- grade tumor using voided urine cytology leads to frequent use of in- vasive cystoscopy. The development of a sensitive noninvasive diagnostic test that could specifically detect bladder carcinoma in the early stages would improve the clinical outcomes by starting the treatment earlier [1–9]. HURP was initially identified through bioinformatics analysis per- formed to identify potentially important regulatory genes of cell cycle. Cells overexpressing HURP appear to have the characteristics of tumor cells with a reduced dependence on extracellular growth factors and the potential to exhibit anchorage independent growth [10–12]. In a previous study, we measured HURP RNA in urine of 211 patients with bladder cancer, 71 benign bladder lesions and 62 normal controls using semi quantitative RT-PCR and we reported 78.7% sensitivity and 94% specificity for bladder cancer detection (unpublished data). Advances in nanotechnology have allowed the development of nano- particle-based assays for sensitive bio-molecular detection [13–17]. Gold nanoparticles (AuNPs) have been used for direct detection of nucleic acids due to their unique optical properties. Colloidal AuNPs are red in color owing to their Surface Plasmon Resonance (SPR) which is the col- lective oscillation of free electrons in metal nanoparticles when electro- magnetic radiation struck the particles. SRP is also responsible for the large absorption and scattering cross-sections of AuNPs which are 4–5 or- ders of magnitude larger than those of conventional dyes [13]. When AuNPs aggregate their color change from red to blue due to a second phenomenon known as plasmon-plasmon transfer [15]. In this study, we developed an AuNP assay for the direct detection of unampli fied HURP RNA in urine samples for diagnosis of bladder cancer. The assay employed MNPs for capturing and purifying the target RNA and AuNPs for detection. The developed HURP AuNP assay was compared to conventional RNA detection by RT-PCR and to the standard diagnostic cystoscopy. Materials and methods Study population The study was approved by Ain Shams Faculty of Medicine ethical committee. A prospective analysis was performed on 100 Egyptian Clinical Biochemistry 47 (2014) 104–110 ⁎ Corresponding author at: YJ-Science & Technology Research Center, Department of Chemistry, The American University in Cairo, SSE, Rm #1184, P.O. Box 74, New Cairo 11835, Egypt. Fax: +20 227957565. E-mail address: hazzazy@aucegypt.edu (H.M.E. Azzazy). 0009-9120/$ – see front matter © 2013 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.clinbiochem.2013.10.022 Contents lists available at ScienceDirect Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem