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 . A lateral flow chromatographic device for detecting the presence of at least one single-stranded target nucleic acid analyte in a fluid sample, comprising a chromatographic test strip which comprises (a) a sample receiving zone for receiving an aliquot of the sample and for receiving a labeled detection oligonucleotide, which detection oligonucleotide comprises a sequence which is complementary to a first sequence of the target nucleic acid; and, (b) a capture zone in lateral flow contact with the sample receiving zone, said capture zone comprising a microporous membrane, onto which at least one capture oligonucleotide is immobilized at a feature size of 500 μm diameter or smaller, and which comprises a sequence which is complementary to a second sequence of the target nucleic acid. 
     
     
         2 . The lateral flow chromatographic device of  claim 1 , wherein the microporous membrane is 3 mm or less in width. 
     
     
         3 . The lateral flow chromatographic device of  claim 1  wherein the sample receiving zone and the capture zone comprise a contiguous microporous membrane. 
     
     
         4 . The lateral flow chromatographic device of  claim 1 , wherein the microporous membrane is a lateral flow compatible nitrocellulose membrane having a pore size of between 0.2 and 20 μm. 
     
     
         5 . The lateral flow chromatographic device of  claim 1 , wherein the detection oligonucleotide is labeled with a detectable particle of between 0.02 and 1 μm in diameter. 
     
     
         6 . The lateral flow chromatographic device of  claim 5 , 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, fluorescently detectable particles, and semi-conductor nanocrystals. 
     
     
         7 . The lateral flow chromatographic device of  claim 1 , wherein the detection oligonucleotide comprises a first portion having a sequence complementary to a part of the target sequence and a second portion having a non-target specific sequence of at least 9 nucleotides, which second portion is adjacent to the label. 
     
     
         8 . The lateral flow chromatographic device of  claim 7 , wherein the second portion has a poly (A) or poly (T) sequence of at least 9 nucleotides. 
     
     
         9 . The lateral flow chromatographic device of  claim 1 , wherein the first sequence and second sequence of the target nucleic acid are adjacent within 2 bases. 
     
     
         10 . The lateral flow chromatographic device of  claim 1 , wherein the capture oligonucleotide is immobilized to the microporous membrane using a non-contact deposition method. 
     
     
         11 . The lateral flow chromatographic device of  claim 1 , wherein the detection oligonucleotide is replaced by a branched nucleic acid molecule or a dendrimeric nucleic acid molecule. 
     
     
         12 . The lateral flow chromatographic device of  claim 1 , wherein the capture oligonucleotide has a feature size of between 50 and 300 μm diameter. 
     
     
         13 . The lateral flow chromatographic device of  claim 1 , wherein the capture oligonucleotide has a feature size of between 50 and 250 μm diameter. 
     
     
         14 . The lateral flow chromatographic device of  claim 1 , wherein the capture oligonucleotide has a feature size of between 50 and 200 μm diameter. 
     
     
         15 . A lateral flow chromatographic device for detecting the presence of at least one single-stranded target nucleic acid analyte in a fluid sample, comprising a lateral flow matrix which defines a flow path and which comprises in series: (a) a sample receiving zone for receiving an aliquot of a fluid sample; (b) a labeling zone in lateral flow contact with said sample receiving zone, wherein the labeling zone comprises a porous material containing at least one detection oligonucleotide reversibly bound thereto, which detection oligonucleotide is complementary to a first sequence of the target nucleic acid and is coupled to a detectable label; and, (c) a capture zone in lateral flow contact with said labeling zone, said capture zone comprising a microporous membrane, onto which at least one capture oligonucleotide is immobilized at a feature size of 500 μm diameter or smaller. 
     
     
         16 . The lateral flow chromatographic device of  claim 15 , wherein the microporous membrane is 3 mm or less in width. 
     
     
         17 . The lateral flow chromatographic device of  claim 15  wherein the sample receiving zone and the capture zone comprise a contiguous microporous membrane. 
     
     
         18 . The lateral flow chromatographic device of  claim 15 , wherein the microporous membrane is a lateral flow compatible nitrocellulose membrane having a pore size of between 0.2 and 20 μm. 
     
     
         19 . The lateral flow chromatographic device of  claim 15 , wherein the detection oligonucleotide is labeled with a detectable particle of between 0.02 and 1 μm in diameter. 
     
     
         20 . The lateral flow chromatographic device of  claim 19 , 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, fluorescently detectable particles, and semi-conductor nanocrystals. 
     
     
         21 . The lateral flow chromatographic device of  claim 15 , wherein the detection oligonucleotide comprises a first portion having a sequence complementary to a part of the target sequence and a second portion having a non-target specific sequence of at least 9 nucleotides, which second portion is adjacent to the label. 
     
     
         22 . The lateral flow chromatographic device of  claim 21 , wherein the second portion has a poly (A) or poly (T) sequence of at least 9 nucleotides. 
     
     
         23 . The lateral flow chromatographic device of  claim 15 , wherein the first sequence and second sequence of the target nucleic acid are adjacent within 2 bases. 
     
     
         24 . The lateral flow chromatographic device of  claim 15 , wherein the capture oligonucleotide is immobilized to the microporous membrane using a non-contact deposition method. 
     
     
         25 . The lateral flow chromatographic device of  claim 15 , wherein the detection oligonucleotide is replaced by a branched nucleic acid molecule or a dendrimeric nucleic acid molecule. 
     
     
         26 . The lateral flow chromatographic device of  claim 15 , wherein the capture oligonucleotide has a feature size of between 50 and 300 μm in diameter. 
     
     
         27 . The lateral flow chromatographic device of  claim 15 , wherein the capture oligonucleotide has a feature size of between 50 and 250 μm in diameter. 
     
     
         28 . The lateral flow chromatographic device of  claim 15 , wherein the capture oligonucleotide has a feature size of between 50 and 200 μm in diameter. 
     
     
         29 . A method of testing for the presence of a target nucleic acid in a liquid sample, comprising applying or contacting the liquid sample to the sample receiving zone of the lateral flow chromatographic device according to  claim 1 , allowing the sample to transport by capillary action through the capture zone, and detecting the presence or absence of the target nucleic acid by detecting the presence of the label at the relevant capture zone feature. 
     
     
         30 . A method for detecting the presence of a target nucleic acid in a biological sample, comprising:
 (a) providing a biological sample suspected of containing the target nucleic acid sequence;   (b) releasing nucleic acid from the biological sample;   (c) amplifying the target nucleic acid using nucleic acid sequence based amplification (NASBA) to generate a solution containing amplified single-stranded RNA complementary to the target nucleic acid, if present in the extracted DNA and/or RNA from the biological sample; and,   (d) assaying for the presence of the complementary RNA target nucleic acid using the method according to  claim 29 .   
     
     
         31 . A kit for testing the presence of a target nucleic acid in a sample, comprising:
 (a) a lateral flow chromatographic device according to  claim 1 ;   (b) a labeled detection oligonucleotide complementary to a second sequence in the target nucleic acid.   
     
     
         32 . A method of testing for the presence of a target nucleic acid in a liquid sample, comprising applying or contacting the liquid sample to the sample receiving zone of the lateral flow chromatographic device according to  claim 15 , allowing the sample to transport by capillary action through the capture zone, and detecting the presence or absence of the target nucleic acid by detecting the presence of the label at the relevant capture zone feature. 
     
     
         33 . A method for detecting the presence of a target nucleic acid in a biological sample, comprising:
 (a) providing a biological sample suspected of containing the target nucleic acid sequence;   (b) releasing nucleic acid from the biological sample;   (c) amplifying the target nucleic acid using nucleic acid sequence based amplification (NASBA) to generate a solution containing amplified single-stranded RNA complementary to the target nucleic acid, if present in the extracted DNA and/or RNA from the biological sample; and,   (d) assaying for the presence of the complementary RNA target nucleic acid using the method according to  claim 32 .   
     
     
         34 . A kit for testing the presence of a target nucleic acid in a sample, comprising:
 (a) a lateral flow chromatographic device according to  claim 15 ;   (b) a labeled detection oligonucleotide complementary to a second sequence in the target nucleic acid.   
     
     
         35 . A lateral flow microarray chromatographic device for detecting the presence or absence of a plurality of single-stranded target nucleic acids in one or more fluid samples, comprising a lateral flow matrix which defines a flow path and which comprises in series:
 (a) a sample receiving zone for receiving the fluid sample(s);   (b) a labeling zone in lateral flow contact with said sample receiving zone, wherein the labeling zone comprises a porous material containing a plurality of different detection oligonucleotides reversibly bound thereto, which detection oligonucleotides are complementary to first sequences of a plurality of respective target nucleic acids and are coupled to detectable labels; and,   (c) a capture zone in lateral flow contact with said labeling zone, said capture zone comprising a microporous membrane, at least a portion of which contains a plurality of different capture oligonucleotides immobilized thereto, which capture oligonucleotides are complementary to second sequences of a plurality of respective target nucleic acids, and wherein the different capture oligonucleotides are immobilized to the microporous membrane at a feature size of 300 μm or less in diameter.   
     
     
         36 . The lateral flow microarray of  claim 35 , wherein the different detection oligonucleotides are coupled to differentiable detectable labels. 
     
     
         37 . The lateral flow microarray of  claim 35 , wherein the differentiable detectable labels are dyed polystyrene microspheres. 
     
     
         38 . The lateral flow microarray of  claim 35 , wherein the differentiable detectable labels are semiconductor nanocrystals with different spectral emission characteristics. 
     
     
         39 . A lateral flow microarray chromatographic device for detecting the presence or absence of a plurality of target nucleic acids in one or more fluid samples, comprising a lateral flow matrix which defines a flow path and which comprises in series:
 (a) a sample receiving zone for receiving the fluid sample(s) and for receiving a plurality of different detection oligonucleotides, each of which detection oligonucleotides comprises a sequence which is complementary to a first sequence of a specific target nucleic acid and is labeled; and,   (b) a capture zone in lateral flow contact with said labeling zone, said capture zone comprising a microporous membrane, at least a portion of which contains a plurality of different capture oligonucleotides immobilized thereto, each of which capture oligonucleotides comprises a sequence which is complementary to second sequence of the specific target nucleic acid, and wherein the different capture oligonucleotides are immobilized to the microporous membrane at a feature size of 300 μm or less in diameter.   
     
     
         40 . The lateral flow microarray of  claim 39 , wherein the different detection oligonucleotides are coupled to differentiable detectable labels. 
     
     
         41 . The lateral flow microarray of  claim 39 , wherein the differentiable detectable labels are dyed polystyrene microspheres. 
     
     
         42 . The lateral flow microarray of  claim 41 , wherein the differentiable detectable labels are semiconductor nanocrystals with different spectral emission characteristics.