Miniaturized lateral flow device for rapid and sensitive detection of proteins or nucleic acids

Abstract

The invention provides miniaturized lateral flow chromatographic and lateral flow chromatographic microarray devices (LFM). The miniaturization of lateral flow nucleic acid detection achieved by the present invention offers reduced reagent use, femtomole sensitivity, excellent linear dynamic range, and rapid detection. Moreover, the small feature sizes of capture oligonucleotides renders the potential information capacity of the platform comparable to more traditional spotted fluorescence microarrays as well as improving sensitivity. The LFM devices exemplified herein enable analytes to be detected within 10 seconds from the time of sample introduction to the LFM device. Sample volumes may be as low as about 10 microliters, significantly reducing assay costs and ameliorating reagent storage logistics. Additionally, the miniaturization of lateral flow opens the door to highly multiplexed assays, allowing many proteins or nucleic acids to be detected in a single assay.

Description

RELATED APPLICATIONS[0001]This patent application claims the benefit of the filing date of U.S. Provisional patent application No. 60 / 839,537 filed Aug. 22, 2006 and Provisional patent application No. 60 / 925,210 filed Apr. 18, 2007 under 35 U.S.C. 119(e).STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]This invention was made with government support under Contract No. DE-AC52-06NA25396, awarded by the United States Department of Energy. The government has certain rights in this invention.BACKGROUND OF THE INVENTION[0003]The threat presented by biological weapons, global heath care issues and emerging diseases of natural origin lend urgency to the development of rapid, field-deployable pathogen detection and diagnostic tools (1,2). Ideally, to be of general field utility, a diagnostic device must be capable of sensitive and specific pathogen detection while retaining simplicity of use and independence from complex laboratory instrumenta...

AI Technical Summary

Benefits of technology

[0009]The LFM technology and LFM devices of the invention offer many of the advantages of microarray technology yet retain the simplicity of lateral flow-based platforms. The miniaturization of lateral flow nucleic acid detection achieved by the present invention offers reduced reagent use, femtomole sensitivity, excellent linear dynamic range, and rapid detection. Moreover, the small feature sizes of capture oligonucleotides renders the potential information capacity of the platform comparable to more traditional spotted fluorescence microarrays as well as improving sensitivity. The LFM devices exemplified herein enable analytes to be detected within 10 seconds from the time of sample introduction to the LFM device. Sample volumes may be as low as about 10 microliters, significantly reducing assay costs and ameliorating reagent storage logistics. Additionally, the miniaturization of lateral flow opens the door to highly multiplexed assays, allowing many proteins or nucleic acids to be detected in a single assay.

[0010]Coupled with an isothermal amplification technique, LFM provides a facile means of rapidly detecting nucleic acid targets while circumventing hardware requirements for fluorescence detection and PCR thermocycling.

[0011]The power of LFM is demonstrated in the Examples, infra. More specifically, Example 8 illustrates the utility of the lateral flow microarray (LFM) approach for sensitive detection and discrimination of closely related microbial signatures when present as minority sequences in complex nucleic acid mixtures, using an assay based on the nonsense mutation in the plcR gene of B. anthracis, that is absent in the near phylogenetic neighbors B. thuringiensis and B. cereus (27,28). The results demonstrate that LFMs, making use of stable detection reagents suitable for dry storage, can be used to detect as little as 250 amol analyte within 2 minutes of sample addition. The miniaturization of lateral flow detection decreases reagent consumption and sample-to-answer times while increasing the potential information capacity of the platform to enable the development of highly multiplexed nucleic acid detection assays.

[0013]In some embodiments, the first sequence and second sequence of the target nucleic acid are adjacent within 2 bases, in order to take advantage of “base stacking” hybridization stability.

[0015]In some embodiments, the first sequence and second sequence of the target nucleic acid are adjacent within 2 bases, in order to take advantage of “base stacking” hybridization stability.

Problems solved by technology

While nucleic acid-based assays for pathogen detection and identification offer sensitivity, specificity and resolution, they are relatively elaborate and often costly, limiting their utility for point-of-care diagnostics and deployment under field conditions where a supporting laboratory infrastructure is limited or absent.

Retaining assay sensitivity, while circumventing requirements for thermocyclers and fluorescence detection hardware, remains a significant challenge.

Nonetheless, the reliance of this technology on costly instrumentation for high-resolution fluorescence signal transduction severely limits the utility of microarrays for field applications where a laboratory infrastructure is limited or unavailable.

Though microarray hybridization times as short as 500 seconds have been reported (9), such methods employ relatively elaborate microfluidic designs that remain reliant upon fluorescent detection and do not address the need for low cost, easily manufactured devices that can be used without costly supporting instrumentation.

Unfortunately, the utility of lateral flow detection in the context of a PCR-based assay is severely limited by the fact that reliance on thermocycling hardware largely negates the potential benefit of the otherwise highly simplified lateral flow platform.

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