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.

1 - 18 . (canceled) 
     
     
         19 . A method for detecting the presence of a target nucleic acid in a liquid sample, comprising:
 a) contacting the liquid sample comprising at least 150 mM salt to a sample receiving zone of a lateral flow device,
 wherein the device comprises: a porous lateral flow strip and a microarray of spots on the porous lateral flow strip, each spot comprising a plurality of capture oligonucleotides, wherein each capture oligonucleotides is complementary to a capture sequence of the target nucleic acid, and wherein each spot was printed with a solution having a concentration of capture oligonucleotides between 200 μM and 800 μM; 
   b) contacting the target nucleic acid with a detection oligonucleotide, wherein the detection oligonucleotide is complementary to a detection sequence of the target nucleic acid;   c) transporting the liquid sample through a capture zone of the device by capillary action; and   d) detecting the presence of the target nucleic acid by detecting the presence of the detection oligonucleotide.   
     
     
         20 . The method of  claim 19 , wherein the lateral flow strip comprises an area of approximately 60 mm 2 . 
     
     
         21 . The method of  claim 19 , wherein each spot has a diameter of between 50 and 300 μm. 
     
     
         22 . The method of  claim 19 , wherein each spot has a diameter of between 50 and 250 μm. 
     
     
         23 . The method of  claim 19 , wherein each spot has a diameter of between 50 and 200 μm. 
     
     
         24 . The method of  claim 19 , wherein the porous lateral flow strip has a pore size of between 0.2 and 20 μm. 
     
     
         25 . The method of  claim 19 , wherein the detection oligonucleotide is a branched nucleic acid molecule or a dendrimeric nucleic acid molecule. 
     
     
         26 . The method of  claim 19 , wherein the detection oligonucleotide is a labeled colorimetric detection oligonucleotide. 
     
     
         27 . The method of  claim 19 , wherein the detection oligonucleotide is labeled with a detectable particle of between 0.02 and 1 μm in diameter. 
     
     
         28 . The method of  claim 27 , 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 semiconductor nanocrystals. 
     
     
         29 . The method of  claim 19 , wherein the detection oligonucleotide comprises a first portion having a sequence complementary to a part of the detection sequence and a second portion having a non-target specific sequence of at least 9 nucleotides, wherein the second portion is adjacent to a label. 
     
     
         30 . The method of  claim 29 , wherein the second portion has a poly (A) or poly (T) sequence of at least 9 nucleotides. 
     
     
         31 . The method of  claim 19 , wherein the capture sequence and detection sequence of the target nucleic acid are adjacent within 2 bases. 
     
     
         32 . The method of  claim 19 , wherein the porous strip comprises lateral flow compatible nitrocellulose. 
     
     
         33 . The method of  claim 32 , wherein the nitrocellulose has pore sizes range between 0.2 and 20 μm. 
     
     
         34 . The method of  claim 19 , wherein detecting the presence of the detection oligonucleotide is by detecting a visual colorimetric signal of the detection oligonucleotide viewable by an unaided human eye. 
     
     
         35 . The method of  claim 19 , wherein the device further comprises a sample pad and/or a conjugate release pad. 
     
     
         36 . The method of  claim 35 , wherein the sample pad and/or the conjugate release pad is impregnated with a substance selected from the group consisting of 4-1(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glucol, SDS, BSA, a hydrophilic polysaccharide, polyvinyl pyrolidone, and combinations thereof.