Magnetization of Hydrogel Particles: a Novel Urine Tuberculosis Antigen Test. L. Paris, A. Cox, Robin Araujo, E. Petricoin, L. Liotta and A. Luchini. George Mason University, 10900 University Blvd, Manassas 20110, VA, lparis2@gmu.edu , alexcox245@gmail.com , raraujo@gmu.edu , epetrico@gmu.edu , lliotta@gmu.edu , aluchini@gmu.edu . ABSTRACT This study aims at developing a nanotechnology-based screening test that would reliably detect early stage tuberculosis (TB) infection and discriminate active from latent infections. The use of urine in TB testing has been hampered by 1) extremely low concentration analytes and 2) protein lability. A novel urine collection system based on bait functionalized hydrogel nanoporous particles is developed that in few instants concentrates the analytes present in a large volume of urine (e.g. 30 mL) in a small volume of bait functionalized particles (e.g. 50 ul) and protects the captured analytes from degradation. Hydrogel particles can be shipped to the analysis laboratory at room temperature without the need of costly freezing preservation. A novel separation method based on magnetic nanoparticle tags permits the harvesting of hydrogel particles in few seconds without using high speed centrifugation. This technology can enable the production of an urinary TB antigen test applicable in developing country settings where the access to conventional laboratory equipment might be limited. Keywords: hydrogel particles, biomarkers, infectious diseases, urine, diagnostics 1 INTRODUCTION A critical unmet goal of the biomedical research sector is the identification of low abundance biomarkers that correlate with therapeutic outcome or toxicity, or that are predictive of early stage disease [1]. Despite a growing interest in the value of biomarkers, the research investment in biomarker discovery and clinical validation has not met the expectations to date [1, 2]. This poor return is due in large part to the low abundance of early disease biomarkers that exist at a concentration below the detection limit of biomarker discovery platforms. Protein biomarker discovery and quantitation by mass spectrometry (MS) and multiple reaction monitoring (MRM) are powerful approaches [1, 2] but are severely limited in their practical application because of technical and physiological constraints [3]: 1) The vast majority of diagnostic analytes measured in the clinical laboratory by immunoassays has a concentration range (50 pg/mL and 10 ng/mL [4]) much lower than the analyte detection sensitivity for MS or MRM applied directly to a complex body fluid (>50 ng/mL) [5]. Thus, the most important protein biomarkers, particularly those derived from early stage disease [6], are invisible to conventional MS or MRM [7]. 2) Proteins and peptides are masked by billion fold excess quantities of resident proteins such as immunoglobulin and albumin. The MS input sample is strictly limited in the maximum total protein (< 50 µg) content, a value lower than the serum or plasma protein content in the microliter volume of the MS input. If the sample is simply concentrated, this will overwhelm the total protein capacity of the MS. 3) candidate biomarkers are very perishable following clinical sample collection. Biomarker instability is a serious problem that has hindered the utility of biospecimens and prevented validation [2]. To overcome these barriers, we created novel nanoporous, buoyant, core-shell hydrogel particles containing novel reactive high affinity chemical baits. In one step following addition to a complex biologic fluid, our particles affinity harvest, preserve, and concentrate desired analytes, while simultaneously excluding unwanted high abundance proteins (Figure 1). Figure 1. Biomarker harvesting: particles carrying affinity bait are mixed in a tube of blood (black As represent albumin and other carrier proteins, red Bs represent biomarkers). When particles are isolated from blood they contain biomarkers of interest that are eluted out of particles and are collected in a smaller volume, yielding high concentration and purification. This new particle technology effectively protected highly labile proteins such as interleukins and growth factors from enzymatic degradation in blood, urine and sweat, and massively increased the effective detection sensitivity, while improving the precision, of multiple reaction monitoring (MRM) analysis [8, 9]. Used in whole blood as a one step, in-solution preprocessing step, the nanoparticles greatly enriched low molecular weight proteins and peptides while excluding unwanted albumin NSTI-Nanotech 2013, www.nsti.org, ISBN 978-1-4822-0586-2 Vol. 3, 2013 69