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.

What is claimed is: 
     
         1 . A method for quantitatively detecting the presence of one or more target nucleic acids in a fluid sample, the method comprising:
 immobilizing first capture oligonucleotides on a first region of a microporous membrane;   immobilizing second capture oligonucleotides on a second region of a microporous membrane;   hybridizing labeled colorimetric detection oligonucleotides to a complementary first sequence of the target nucleic acid;   directly hybridizing the first capture oligonucleotides to a complementary second sequence of a first group of the target nucleic acids;   directly hybridizing the second capture oligonucleotides to a complementary third sequence of a second group of the target nucleic acids, wherein the first capture oligonucleotides and the second capture oligonucleotides each display signal linearity over a different concentration range of the target nucleic acid;   measuring an intensity of a first optical or colorimetric signal produced on the first region;   measuring an intensity of a second optical or colorimetric signal produced on the second region;   determining if the intensity of the first signal and/or the second signal is in the linear range of the first capture oligonucleotide and second capture oligonucleotide respectively; and   calculating the concentration of the target nucleic acid in the fluid sample using the intensity of the first signal and/or the second signal if the intensity is in the respective linear range.   
     
     
         2 . The method of  claim 1  wherein the microporous membrane comprises lateral flow compatible nitrocellulose. 
     
     
         3 . The method of  claim 1  wherein the detection oligonucleotide is labeled with a detectable particle of between 0.02 and 1 μm in diameter. 
     
     
         4 . The method of  claim 3  wherein the detectable particle is selected from the group consisting of polystyrene microspheres, latex particles, nano-gold particles, colloidal gold particles, metal particles, magnetic particles, and semi-conductor nanocrystals. 
     
     
         5 . The method of  claim 1  wherein the detection oligonucleotides each comprise a first portion having a sequence complementary to the first sequence and a second portion having a non-target specific sequence of at least 9 nucleotides, which second portion is adjacent to the label. 
     
     
         6 . The method of  claim 5  wherein the second portion has a poly (A) or poly (T) sequence of at least 9 nucleotides. 
     
     
         7 . The method of  claim 1  wherein the first sequence and second sequence of the target nucleic acid are adjacent within 2 bases. 
     
     
         8 . The method of  claim 1  wherein each detection oligonucleotide is a branched nucleic acid molecule or a dendrimeric nucleic acid molecule. 
     
     
         9 . The method of  claim 1  wherein each first capture oligonucleotide and/or second capture oligonucleotide has a feature size of between 50 and 300 μm diameter. 
     
     
         10 . The method of  claim 9  wherein each first capture oligonucleotide and/or second capture oligonucleotide has a feature size of between 50 and 250 μm diameter. 
     
     
         11 . The method of  claim 10  wherein each of the first capture oligonucleotides and/or second capture oligonucleotides has a feature size of between 50 and 200 μm diameter. 
     
     
         12 . The method of  claim 1  further comprising:
 releasing nucleic acids from a biological sample, the nucleic acids suspected of containing the target nucleic acid sequence; and 
 amplifying the target nucleic acid sequence to produce copies of the target nucleic acid. 
 
     
     
         13 . The method of  claim 12  wherein the copies of the target nucleic acid comprise DNA or RNA. 
     
     
         14 . The method of  claim 12  wherein the amplifying step comprises reverse transcription polymerase chain reaction (RT-PCR) or nucleic acid sequence based amplification (NASBA). 
     
     
         15 . The method of  claim 1  comprising hybridizing different labeled colorimetric detection oligonucleotides to different target nucleic acids. 
     
     
         16 . The method of  claim 15  wherein the different detection oligonucleotides are coupled to differentiable detectable labels. 
     
     
         17 . The method of  claim 16  wherein the differentiable detectable labels comprise dyed polystyrene microspheres. 
     
     
         18 . The method of  claim 16  wherein the differentiable detectable labels comprise semiconductor nanocrystals with different spectral emission characteristics.