Microfluidic System for Solution Array Based Bioassays

The objective of this project is to demonstrate new enabling technology for multiplex biodetection systems that are flexible, miniaturizable, highly automated, low cost, and high performance. It builds on prior successes at LLNL with particle-based solution arrays, such as those used in the Autonomous Pathogen Detection System (APDS) successfully field deployed to multiple locations nationwide. We report the development of a multiplex solution array immunoassay based upon engineered metallic nanorod particles. Nanobarcodes® particles are fabricated by sequential electrodeposition of dissimilar metals within porous alumina templates, yielding optically encoded striping patterns that can be read using standard laboratory microscope optics and PC-based image processing software. The addition of self-assembled monolayer (SAM) coatings and target-specific antibodies allows each encoded class of nanorod particles to be directed against a different antigen target. A prototype assay panel directed against bacterial, viral, and soluble protein targets demonstrates simultaneous detection at sensitivities comparable to state of the art immunoassays, with minimal cross-reactivity. Studies have been performed to characterize the colloidal properties (zeta potential) of the suspended nanorod particles as a function of pH, the ionic strength of the suspending solution, and surface functionalization state. Additional studies have produced means for the non-contact manipulation of the particles, including the insertion of magnetic nickel stripes within the encoding pattern, and control via externally applied electromagnetic fields. Using the results of these studies, the novel Nanobarcodes® based assay was implemented in a prototype automated system with the sample processing functions and optical readout performed on a microfluidic card. The unique physical properties of the nanorod particles enable the development of integrated microfluidic systems for biodefense, protein expression studies, and other applications. Introduction/Background Multiplex bioassays are critical for biodefense and medical diagnostic applications. In many cases, multiple threat agents must be screened simultaneously, or multiple samples must be rapidly tested [1]. Host-response assays require detection and monitoring of combinations of biomarkers, including DNA, RNA, proteins, and small molecules. While tremendous advances in multiplexed biomarker analysis have come about with the advent of DNA and protein chips, this technology is limited in several important ways (see Table I for a comparison between solution arrays and chip technology): (1) microarrays are 2-dimensional, limiting interaction with target molecules; (2) gene and protein chips are aimed at only one type of marker (DNA, RNA, or protein); (3) DNA and protein chips are not reconfigurable; and (4) microarray technologies require the manufacture of specific chips, an approach that is expensive and time consuming. Solution arrays address all of these shortcomings.