The diverse nature of biological sample matrices present a need for robust yet general front-end sample processing methods that enable the collection of trace analytes even when present in complex mixtures of non-probative sample constituents. These challenges are often compounded by the presence of materials confounding to effective immunological or molecular analytical techniques. For example, samples derived from human tissue are likely to contain complex polysaccharides, hemoglobin, iron and other substances known to be inhibitory to DNA polymerases employed for polymerase chain reaction (PCR). Similarly, environmental samples or trace samples contaminated with environmental constituents, such as soil or plant material, can also contain organic materials, such as humic acids, that are strongly inhibitory to PCR and other enzymatic reactions critical to thorough nucleic acid analysis.
Although reliable nucleic acid isolation methods applicable to diverse biological samples have been reported for both DNA and RNA, such methods are labor intensive, dependent upon laboratory instrumentation and require hours to complete resulting in limited sample throughput and significant sample backlogs. Down-stream enzymatic manipulations, such as polymerase chain reaction (PCR), can be adversely impacted by the presence of matrix constituents inhibitory to enzymatic activity rendering reliable sample preparation indispensable. Hemoglobin, iron and complex polysaccharides are commonly encountered in biological samples while additional inhibitory compounds such as humic acids often accompany environmentally collected samples containing soil, plant material or decaying mater. Additionally, the trace nature of many analytes in diagnostic and forensic samples as well as the abundance of closely related but non-probative constituents contribute significantly to analytical challenges.
Lateral flow immuno-chromatography is well established and has been used for the detection of proteins and small molecules for many years. Indeed, immuno-capture during lateral flow is the basis for rapid hand-held immuno-assays that have found widespread use in the point-of-care (e.g. group A Streptococcal antigen) and in the home (e.g. pregnancy tests). While these assays make use of immuno-capture during lateral flow as a detection end-point, we propose the use of the same principle as a means of attaining rapid and efficient immuno-capture as a first step in a sample preparation strategy designed to enable the recovery of scarce targets (cells, viruses, spores) from mixed samples. Once captured in the stationary phase, these targets can then be subjected to further processing for nucleic acid isolation or collected for other analyses.
Nucleic acid-based assays for pathogen detection and identification offer sensitivity, specificity and resolution. These characteristics render nucleic acid analysis a powerful diagnostic and forensic technique. Nonetheless, many technologies for nucleic acid preparation have focused on isolation from relatively abundant samples such as clinical blood specimens. Many applications, however, often must address the need to isolate and identify trace constituents in complex mixed samples of diverse origin. In contrast to DNA-based assays, immunoassays have found widespread acceptance in low cost, easily used formats, perhaps most notable of which is the chromatographic lateral flow immunoassay. Lateral flow assays, also known as hand-held assays or dipstick assays, are used for a broad range of applications where rapid antigen detection is required in an easily used, low cost format. Lateral flow immunoassays have been successfully employed for pathogen identification, diagnostics, and environmental and agriculture surveillance. Several chromatographic lateral flow assays have been described for the detection of nucleic acid sequences using a variety of detection techniques. Early work made use of cumbersome enzymatic detection strategies that relied on time consuming manipulations of dipsticks following introduction of the sample and detection schemes poorly suited for multiplexed applications.
More recently described, the Lateral Flow Microarray (LFM) is a miniaturized lateral flow-based method for multiplexed nucleic acid detection (Carter, D. J. and R. B. Cary, Lateral flow microarrays: a novel platform for rapid nucleic acid detection based on miniaturized lateral flow chromatography. Nucleic Acids Res, 2007. 35(10): p. e74). The approach makes use of DNA microarray-like patterning of a small lateral flow chromatography strip allowing multiple nucleic acid sequences to be detected in a single assay. The reduced surface area of the device confers several advantages over traditional lateral flow device form factors. Sample volumes are reduced to 10 μL resulting in reduced reagent consumption as well as reduced sample transport times. Moreover, hybridization times exhibited by the lateral flow microarray (LFM) are significantly reduced compared to standard glass substrate microarrays, which typically are allowed to hybridize with sample for several hours, as well as more complex microarray implementations that make use of microfluidic systems to facilitate more rapid hybridization. The convective fluid movement through the lateral flow substrate as well as the open-ended pores of the membrane substrates employed result in superior chromatography performance compared to bead-based column chromatography. These factors result in hybridization-based detection of <250 amol of analyte in 2 minutes. LFM is further described in U.S. patent application Ser. No. 11/894,910 and PCT International Application No. PCT/US2007/018537.
The LFM platform has been used to develop a rapid assay for Bacillus anthracis, the causative agent of anthrax, and has been shown to detect RNA from as few as 2-3 B. anthracis cells when present in a complex nucleic acid background consisting of 1 μg of total human RNA. The reported LFM approach made use of standard laboratory methods for RNA isolation and an isothermal RNA amplification scheme known as nucleic acid sequence based amplification (NASBA). Perhaps most significantly, the miniaturization of lateral flow exemplified by the LFM offers a physical configuration amenable to integration with fluidic or microfluidic systems for sample preparation support.
Integration of LFM-based protein and nucleic acid detection with simplified sample processing methods would offer several potential advantages for processing and screening of a broad range of sample types, and is desirable. Similarly, more robust sample preparation methods applicable to trace and/or dilute analytes would greatly facilitate nucleic acid amplification and detection in point of care and field deployed assays.