Miniaturized Lateral Flow Device for Rapid and Sensitive Detection of Proteins or Nucleic Acids
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.
This patent application is a continuation of U.S. patent application Ser. No. 11/894,910, entitled “Miniaturized Lateral Flow Device for Rapid and Sensitive Detection of Proteins or Nucleic Acids”, filed Aug. 22, 2007, which claims the benefit of the filing date of U.S. Provisional Patent Application No. 60/839,537 filed Aug. 22, 2006 and U.S. 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 DEVELOPMENTThis 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 INVENTIONThe threat presented by biological weapons, global health 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 instrumentation (3). Additional challenges are presented by the need to screen samples for multiple pathogenic or toxic agents, a characteristic highly desirable in cases where commonalities in early symptom presentation confound differential diagnoses.
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. Reliance upon polymerase chain reaction (PCR) and fluorescent detection of amplified nucleic acids has contributed significantly to the complexity and cost of nucleic acid diagnostics (2,4-6). Retaining assay sensitivity, while circumventing requirements for thermocyclers and fluorescence detection hardware, remains a significant challenge.
The recent advent of DNA microarray technology has promised to increase the information capacity of nucleic acid diagnostics and enable the highly multiplexed detection of genetic signatures (7). The potential of DNA microarrays to detect, in parallel, large panels of distinct nucleic acid sequences has proven to be a powerful technique for many laboratory applications (for review see (8)). 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. Additionally, the long hybridization incubations required for microarray assays increase sample-to-answer times beyond what would be acceptable for a rapid screening assay. 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.
In contrast to DNA-based assays, immunoassays have found widespread acceptance in low cost, easily used formats, perhaps the most notable of which is the chromatographic lateral flow immunoassay (for a review see (10)). 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. Expanding the domain of lateral flow chromatography to nucleic acid detection, a number of recent reports have described lateral flow detection of PCR products using a variety of capture and detection schemes (11-14). 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. Additionally, a PCR-based approach to lateral flow detection necessitates each PCR reaction be subjected to post-amplification manipulations required to generate single-stranded products for hybridization-based detection.
Recent work has sought to alleviate reliance on PCR through employing isothermal nucleic acid amplification schemes or direct detection of unamplified genetic material. Enabled by the use of up-converting phosphor reporters, unamplified Streptococcus pneumoniae DNA sequence has been detected using a lateral flow assay format (15). Up-converting phosphor technology, while sensitive, remains dependent upon the hardware required to detect phosphor emission (16). The use of simple colorimetric detection schemes that circumvent the requirements for complex instrumentation require an upstream amplification strategy to attain suitable sensitivity. Isothermal nucleic acid amplification coupled with lateral flow detection has been reported for assays making use of cycling probe technology (CPT, (17)) and nucleic acid sequence-based amplification (NASBA, (18-20)) (21-25). While the work by Fong et al (21) made use of a lateral flow immuno-assay for DNA detection, the RNA targets amplified by NASBA in the work from Baeumner's group (22-25) were detected using a lateral flow system enabled by the use of liposome encapsulated dye and a sandwich hybridization assay similar to that reported by Rule et al (12). While shown to display nanomolar sensitivity, the reported dye encapsulating liposome-based methods require additional washing steps and the liposomes are relatively labile, must be custom synthesized, and stored under stabilizing hydrated conditions (26).
SUMMARY OF THE INVENTIONThe invention provides miniaturized lateral flow chromatographic and lateral flow chromatographic microarray devices (collectively, “LFM devices”), also termed “DNA dipstick”, “nucleic acid dipstick”, LFM dipstick” and “dipstick” devices, as well as diagnostic assay methods utilizing LFM technology and dipsticks and related diagnostic kits comprising LFM dipsticks.
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.
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.
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.
In one aspect, the invention provides 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. In some embodiments, the microporous membrane is 3 mm or less in width. The lateral flow chromatographic device may combine the sample receiving zone and the capture zone, such that they comprise a contiguous microporous membrane. The microporous membrane is a lateral flow compatible nitrocellulose membrane having a pore size of between 0.2 and 20 μm. The detection oligonucleotide is labeled with a detectable particle of between 0.02 and 1 μm in diameter, including without limitation, polystyrene microspheres, latex particles, nano-gold particles, colloidal gold particles, metal particles, magnetic particles, fluorescently detectable particles, and semi-conductor nanocrystals. In some embodiments, 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. The second portion may, for example, be a poly (A) or poly (T) sequence of at least 9 nucleotides.
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.
In another aspect, the invention provides 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. In some embodiments, the microporous membrane is 3 mm or less in width. The lateral flow chromatographic device may combine the sample receiving zone and the capture zone, such that they comprise a contiguous microporous membrane. The microporous membrane is a lateral flow compatible nitrocellulose membrane having a pore size of between 0.2 and 20 μm. The detection oligonucleotide is labeled with a detectable particle of between 0.02 and 1 μm in diameter, including without limitation, polystyrene microspheres, latex particles, nano-gold particles, colloidal gold particles, metal particles, magnetic particles, fluorescently detectable particles, and semi-conductor nanocrystals. In some embodiments, 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. The second portion may, for example, be a poly (A) or poly (T) sequence of at least 9 nucleotides.
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.
In another aspect, the invention provides an assay 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 of the invention, 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.
In another aspect, the invention provides 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 assay method above.
Unless otherwise defined, all terms of art, notations and other scientific terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this invention pertains, unless otherwise defined. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not be construed to represent a substantial difference over what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and commonly employed using conventional methodologies by those skilled in the art, such as, for example, the widely utilized molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd. edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Current Protocols in Molecular Biology (Ausbel et al., eds., John Wiley & Sons, Inc. 2001. As appropriate, procedures involving the use of commercially available kits and reagents are generally carried out in accordance with manufacturer defined protocols and/or parameters unless otherwise noted.
Overview of LFM System
The invention relates to miniaturized lateral flow chromatographic methods and devices useful for the sensitive and specific detection of nucleic acid and protein analytes. As the invention realizes many of the benefits of microarray technology, incorporated into a lateral flow technology platform, the term “lateral flow microarray” or “LFM” is used herein. Principal features of LFM include small feature sizes (spot sizes) compared to traditional lateral flow devices (i.e., typically less than 600 μm diameter, more typically less than 300 μm diameter, and in some embodiments, smaller, i.e., 50 μm diameter or less), reduced width of the microporous detection membrane, high feature density potential, and multiplex capability. These features, in turn, result in lower sample volume requirements (i.e., 10 μL), faster assay run times, lower reagent costs, and surprising levels of sensitivity (i.e., attomolar) and linear dynamic range (i.e., 3 orders of magnitude signal linearity).
The sensitivity of lateral flow nucleic acid detection methods previously reported in the literature has been on the order of 1 fmol (e.g., (25)). In embodiments of LFM which utilize dyed polystyrene microspheres as the detection particle (i.e., colorimetric detection), the LFM platform provides rapid detection of as little as 250 amol of target using a low cost and widely available flatbed scanner, a standard personal computer system and a commercially available microarray data extraction suit or free image analysis software. This detection limit is similar to the sensitivity reported for fluorescence and chemiluminescence microarray detection strategies (9,46). Furthermore, it is likely that the sensitivity of LFM may be improved by using semi-conductor nanocrystal as the detection particle. Importantly, utilizing nanocrystals in LFM assays results in improved linear dynamic range (see Examples, infra).
LFM demonstrates excellent linear dynamic range utilizing dyed microspheres as the detection particle. Indeed, with reference to the studies described in Example 6, infra, the effective linear range of the LFM assay extends over a 400-fold range of target from 250 amol to 100 fmol (see
Studies utilizing semi-conductor nanocrystals as the detection particle indicate that the linear dynamic range of LFM may be improved to at least three orders of magnitude. As shown in Example 8, remarkable signal linearity over the 1 fmol to 1000 fmol range of dnaR89 analyte was achieved using LFM devices containing nanocrystal-conjugated detection oligonucleotides (R2=0.991).
LFMs offer several advantages arising directly from the miniaturization of the system without sacrificing detection sensitivity. While traditional lateral flow assays make use of sample volumes on the order of hundreds of microliters to milliliters, the miniaturization approach embodied in the invention reduces sample volume to about 10 μl. This reduced sample volume significantly decreases the consumption of reagents required for amplification, and thus assay cost. In the Examples disclosed herein, the LFM device enabled a reduction in the standard NASBA reaction volumes from 20 μl to 2 μl, thereby achieving a one order of magnitude reduction in enzyme consumption. Similarly, other amplification schemes, such as those that make use of microfluidic systems or lab-on-a-chip technologies, may be integrated with LFM-based detection systems to provide a rapid and cost effective means of detecting analytes.
A further benefit of LFM is the time required to detect analyte following introduction of amplified sample material. While the procedures used in Example 8 employed NASBA amplification and traditional RNA isolation protocols requiring approximately 90 minutes to complete, more recent advances in nucleic acid preparation and amplification have reported significant reduction in sample processing times (for a recent review see (47)). As amplification protocols become more rapid, the speed with which amplicons can be detected, without reliance on complex optical systems and fluorescent detection methods, will be critical to realizing the potential of these technologies. The LFM methods of the invention are able to achieve detection of nucleic acid analytes in less than 2 minutes. Given that 250 amol is equivalent to 1.5×108 molecules, efficient amplification methods that offer 109 fold amplification, widely cited amplification levels for PCR- and NASBA-based techniques (22,48), would theoretically enable the detection of single copy targets by LFM following amplification. Future systems that couple advanced amplification technologies and compatible streamlined nucleic acid preparation modalities with rapid LFM detection will allow significant decreases in sample-to-answer times without costly or complex instrumentation.
LFM devices of the invention utilize sandwich-type hybridization, either employing sets of target-complementary oligonucleotides (or other nucleic acid molecules, such as dendrimers) to detect nucleic acid analytes, or binding ligands such as antibodies to detect protein analytes. In respect of nucleic acid detection methods using LFM, nucleic acid target is detected redundantly using (a) detectably labeled detection oligonucleotides complementary to one of two signature sequences on the target nucleic acid (i.e., oligonucleotides conjugated to a detectable label, such as dyed microspheres, semi-conductor nanocrystals, etc.), and (b) membrane-immobilized capture oligonucleotides complementary to the other signature sequence on the target. In the practice of a nucleic acid detection assay utilizing the LFM system of the invention, the capture of amplified target nucleic acids by the membrane-immobilized capture oligonucleotides and labeled detection oligonucleotides brings the label into contact with the membrane, displaying a visual or machine-readable optical signal. Thus, the assay requires positive hybridization to two distinct sequences on the target nucleic acid in order to produce a localized signal, resulting in very high assay specificity.
Physical Components of LFM Devices
The lateral flow chromatographic devices of the invention comprise a series of absorbent substrates which are used to transport analyte in a lateral manner to components containing certain reagents or materials required for the detection of the analyte.
In one aspect, a lateral flow chromatographic device of the invention comprises 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 and which comprises a sequence which is complementary to a second sequence of the target nucleic acid. In an alternative embodiment, a labeling zone in lateral flow contact with said sample receiving zone is inserted up-stream of the capture zone and is lateral flow contact with the capture zone. A 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, thereby enabling the label step to take place on the device.
In a simplified illustration, one embodiment of the LFM device is structurally organized into at least 3 zones, comprising in linear orientation: (a) a sample pad constructed from absorbent material onto which a liquid, nucleic acid-containing sample is deposited, (b) a conjugate release pad containing a least one oligonucleotide-fitted detection particle (e.g., microsphere, bead, quantum dot), and (c) a detection zone comprising a nitrocellulose or nylon membrane containing at least one immobilized capture oligonucleotide. In some embodiments, a fourth element comprises an absorbent material which is capable of facilitating the lateral flow of the liquid sample from the sample pad end of the device to and through the detection zone. In some embodiments, the sample pad (a) and the conjugate release pad (b) are combined. In alternative embodiments, the conjugate release pad element is eliminated, and the sample to be assayed for the presence of a target nucleic acid is mixed with the oligonucleotide-fitted detection particle prior to placing the sample onto the sample pad.
The first substrate, or sample pad or sample receiving zone, comprises an absorbent material preferably composed of a matrix, with minimal nucleic acid binding properties, that will permit unobstructed migration of the nucleic acid analyte to subsequent stages of the apparatus without depletion. In a specific embodiment, the sample pad is composed of a cellulose fiber pad such as Millipore cellulose fiber sample pad material (Cat #CFSP223000).
In embodiments where separate sample and conjugate release pads are employed in the LFM device, the sample pad is situated within the device such that it is in physical contact with the conjugate release pad.
The substrate which contains the labeled detection oligonucleotide conjugate is termed the conjugate release pad or labeling zone. In some embodiments, the labeling zone is also used to receive sample directly. The conjugate release pad comprises a matrix composed of a material with minimal nucleic acid binding capacity and of a physical composition which allows dried detection particles to be liberated into solution with minimal residual binding to the matrix. Examples of materials suitable for conjugate pads include glass fiber and polyester materials (e.g., rayon). These materials are commonly available from various commercial sources (e.g., Millipore, Schleicher & Schuell).
The detection membrane of the capture zone may be any microporous membrane material which is lateral flow compatible, typically microporous cellulose or cellulose-derived materials such as nitrocellulose (e.g., HiFlow 135, Millipore) or nylon. In some embodiments, the sample receiving zone and the capture zone comprise a contiguous microporous membrane.
Typically, the microporous membrane defines a relatively narrow flow path. This may be achieved, for example, by utilizing narrow strips of microporous membrane material. Excellent results are obtained with membrane strips of 5 mm or less in width, with the best results being obtained with strips of 3 mm or less. As will be appreciated, other means for retaining a narrow flow path of less than 5 mm or less than mm may be used, and may include without limitation the use of barriers which define borders which limit the flow path to a channel.
The microporous membrane of the capture zone is a lateral flow compatible membrane such as cellulose, nitrocellulose, polyethersulfone, polyvinylidine fluoride, nylon, charge-modified nylon, and polytetrafluoroethylene. Typically, the membrane is nitrocellulose. The detection membrane is typically provided with a backing material for support, such as mylar or similar plastic materials. The membrane may be treated with agents that inhibit non-specific binding of analyte or other reagents used in an LFM assay.
In embodiments utilizing nitrocellulose, pore sizes typically range between 0.2 and 20 μm, and more typically between 0.2 and 12 μm. In preferred embodiments utilizing particle labels, the pore size of the microporous membrane should be on the order to about 10 times the diameter of the particle.
In one embodiment, the detection membrane is composed of a supported nitrocellulose membrane of sufficiently large pore structure to allow the unimpeded transport of detection reagent through the membrane matrix. Examples of suitable nitrocellulose materials for dyed microsphere mediated detection are Millipore HiFlow Plus HF09004, HF13504, Schleicher & Schuell Prima 60, Schleicher & Schuell Prima 85. The Millipore HF13504 nitrocellulose membrane has been demonstrated to provide rapid, specific and sensitive detection when patterned with appropriate capture oligonucleotides (see Examples, infra). The microporous membrane is placed in lateral flow contact with the labeling zone (conjugate release pad).
In some embodiments, an absorbent material is placed in lateral flow contact with the distal end of the detection membrane in order to facilitate lateral flow through the entire LFM device. Materials suitable for use as an absorbent pad include any absorbent material, including, but not limited to, nitrocellulose, cellulose esters, glass (e.g., borosilicate glass fiber), polyethersulfone, cotton, dehydrated polyacrylamide, silica gel, and polyethylene glycols. The rate of capillary flow can be controlled by choosing the appropriate absorbent zone material.
LFM devices may be encased in a housing as described in, e.g., U.S. Pat. No. 5,451,504. Materials for use in the housing include, but are not limited to, transparent tape, plastic film, plastic, glass, metal and the like. Such housings preferably contain an opening or sample port for introducing sample, as well as a window(s) permitting the visualization of the detection zone(s) of the detection membrane.
Microarray Fabrication:
In the fabrication of an LFM device, the microporous membrane of the capture zone is used for patterning capture oligonucleotides and or protein capture ligands (i.e., antibodies).
In preferred nucleic acid detection LFM fabrications, capture oligonucleotides are patterned onto the detection membrane or substrate (i.e., nitrocellulose) with spot diameter sizes (“feature sizes”) of about 1 mm or less, preferably 600 μm or less, more preferably less than about 300 μm diameter, and in some embodiments, smaller (i.e., 50 to 200 μm, 50 to 250 μm, 50 to 300 μm). Oligonucleotide concentrations for spotting are generally in the range of 200 μM-800 μM. In embodiments in which PNAs or LNAs are used to synthesize oligonucleotides, lower densities may suffice.
Detection membranes may be patterned to suit the desired design of the detection element of the device. Methods for depositing nucleic acids and proteins onto microporous membranes such as nitrocellulose are well know, and negative and positive control reagents as well as capture reagents may be patterned on to the detection membrane using any of a number of deposition techniques. These techniques can be selected based on the density of information to be represented on the detection membrane. Manual deposition by pipette, automated deposition by robotics through contact mediated processes (stainless steel pins on a contact microarray printing robot) or noncontact mediated processes such as piezo responsive micropipettes, may all be used successfully to fabricate the nucleic acid detection device described here.
Preferably, when using nitrocellulose and similar membranes, non-contact printing techniques are used to deposit capture oligonucleotides or proteins onto the detection membrane, in order to retain the structural integrity of the detection membrane material. See. For example, the non-contact printing methods utilized in the Examples which follow.
Additionally, more convention means may be employed, including various techniques commonly used to fabricate hand held assay devices for the immunological detection of proteinaceous analytes in the context of a lateral flow immunochromatographic device.
For example, immobilization of capture oligonucleotides directly on the detection membrane may be accomplished by using high salt to adsorb the nucleic acid molecules to the surface of the membrane, combined with baking at about 80° C. to permanently fix the adsorbed oligonucleotides. Additionally, oligonucleotides may be deposited onto the membrane (i.e., nitrocellulose), air dried, and subjected to UV radiation (see Examples herein). Capture oligonucleotides may also be fixed directly to detection membrane by vacuum transfer in the presence of an equimolar concentration of sodium chloride and sodium citrate, or by the use of ultraviolet irradiation. The capture oligonucleotides may also be covalently linked to charge-modified nylon. In other embodiments, capture oligonucleotides may incorporate a reactive ligand (e.g., biotin) and may be immobilized indirectly on the detection membrane as a result of the interaction between the ligand and an immobilized member of a binding pair (e.g., streptavidin).
Detection membranes may be patterned with positive and negative control reagents and capture reagents in an array such that the physical position of each reagent is known. Positive control reagents can be composed of oligonucleotides complementary to detection oligonucleotides immobilized on detection reagents (i.e. dyed microspheres linked to oligonucleotides through a covalent bond or through an affinity interaction such as that mediated by streptavidin/biotin interactions). Alternatively, in embodiments where the streptavidin/biotin interaction is used to couple dyed microspheres to oligonucleotides the positive control array element can be composed of biotin in any of a number of forms suitable for immobilization on nitrocellulose (for example, a biotin labeled nucleic acid). Following binding to detection oligonucleotides, free biotin binding sites on streptavidin-conjugated dyed microspheres remain available for interaction with immobilized biotin on the detection membrane, thus providing one form of positive control.
Another positive control may be achieved by the immobilization of oligonucleotide on the detection membrane. The use of an oligonucleotide complementary to the dyed microsphere-conjugated detection oligonucleotide as a positive control allows direct hybridization of the detection oligonucleotide/dyed microsphere complex following lateral flow chromatography over the positive control. Negative controls for hybridization specificity can be incorporated into the device by patterning the detection membrane with detection oligonucleotide or other nucleic acid sequences predicted, by means known to those skilled in the art, to not hybridize to the detection oligonucleotide sequence.
For nucleic acid analytes, capture reagents are composed of oligonucleotides synthesized such that the sequence is complementary to a region of the analyte target nucleic acid not overlapping with the region complementary to the detection oligonucleotide. Ideally, the predicted secondary structure of the analyte target nucleic acid is examined to identify those regions exhibiting reduced likelihood of participating in intramolecular hydrogen bonds. Such regions are preferable sites for detection and capture oligonucleotide binding.
Array elements may take the form of lines, stripes, dots or human readable icons, letters or other forms or shapes deemed useful to the interpretation of device read-out. In the case of spots or dots deposited by robotic or manual means, individual feature sizes from 50 microns to 5 mm have been shown to provide accurate and interpretable hybridization mediated detection of 20 fmol analyte DNA molecules.
Capture and Detection Oligonucleotides:
For nucleic acid analytes, LFM devices incorporate two classes of oligonucleotide referred to here as capture and detection oligonucleotides. The detection oligonucleotide is linked by any of a number of means to a detection reagent or label that, when concentrated by capture through hybridization, renders the capture zone distinguishable (i.e., opically) from the surrounding substrate and from additional capture zones where the detection reagent has not been sequestered. Examples of detection reagents include polystyrene microspheres, latex particles, nano-gold particles, colloidal gold particles, metal particles, magnetic particles, fluorescently detectable particles, and semi-conductor nanocrystals and the like.
Alternatively, a nucleic acid complex, such as a DNA dendrimer or branched-DNA molecule, carrying multiple detectable moieties, such as fluorescent molecules or biotin, can be used to amplify lateral flow microarray signal intensity. By generating DNA dendrimers carrying a detection sequence complementary to a region of the target (detection sequence) each hybridization event at the LFM capture zone results in the localization of multiple detectable labels. Using a highly biotinylated dendrimer and a streptavidin conjugated detection particle such as a dyed microspheres or semi-conductor nanocrystals, both colorimetric and fluorescent signal amplification can be realized. For example, the large number of streptavidin binding sites on biotinylated dendrimers will increase the number of streptavidin bound particles captured by each hybridization event and generate a correspondingly amplified signal. Several potential advantages, especially with respect to multiplexed detection, may be realized using this approach. Specifically, the use of a generic biotin/streptavidin interaction allows the simultaneous use of multiple detection probe sequences without requiring the preparation of multiple quantum dot-detection probe conjugates. Together with the use of generic tag sequences added to amplicons through the use of specially designed NASBA primers, this approach is compatible with the development of generic tag-based LFMs suitable for the detection of differing panels of pathogens without redesign of the LFM layout.
The detection oligonucleotide is designed such that the melting temperature of the resulting oligonucleotide allows hybridization to its cognate sequence on the analyte under ambient conditions with sufficient rapidity to allow duplex formation to occur during lateral flow. Detection oligonucleotides with Tm of 50-70° C. have been shown to provide effective reagents for the detection of relevant analytes (using approximately 20-mer oligonucleotides).
Detection oligonucleotides are synthesized with suitable modifications to allow the efficient linkage to appropriate detection reagent. In some embodiments it is advantageous to include a spacer sequence consisting of 9 to 20 T residues proximal to the modified end of the oligonucleotide that will be coupled to the detection reagent. Chemistries of known suitability for use in the device include biotin/streptavidin through a biotin incorporated onto either the 5′ or 3′ end of the detection oligonucleotide and covalent cross-linking through a primary amine incorporated into either the 3′ of 5′ end of the detection oligonucleotide. In one preferred process, detection oligonucleotides are covalently linked to polystyrene microspheres using the coupling agent 1-etyl-3-(3-dimethylaminopropyl-diimide HCl (EDAC). Other methods that mediate the formation of a stable complex between the detection reagent and the detection oligonucleotide under assay conditions should also be suitable for use in the fabrication of the device.
The second class of oligonucleotide used in the device is the capture oligonucleotide. This reagent is immobilized on the microporous detection membrane through the use of standard methods for coupling nucleic acids to nitrocellulose or nylon, including without limitation drying followed by ultraviolet light cross-linking using 0.5 Joules UV. The capture oligonucleotide is designed such that the sequence is complementary to the analyte target nucleic acid at a region predicted to have little or no secondary structure. The length of the capture oligonucleotide is typically approximately 20 bases in length or of a length, to generate a predicted melting temperature of approximately 50-70° C.
In some embodiments it may be advantageous to add a spacer sequence consisting of 9 to 20 T residues.
Detection and capture oligonucleotides can be synthesized using well known DNA synthesis chemistries. The incorporation of modified nucleic acids such as PNA (peptide nucleic acid) or LNA (locked nucleic acid) may be useful for the enhanced hybridization properties of these DNA derivatives. The use of PNA or LNA moieties in the preparation of detection and/or capture oligonucleotides will be useful in manipulating the desired melting temperature, and so may allow shorter oligonucleotides to be employed for detection and/or capture where sequence constraints preclude longer DNA oligonucleotides.
In some embodiments, detection and capture oligonucleotides are designed to hybridize to target nucleic acid within 0, 1 or 2 bases of each other, in order to increase the stability of hybridization via the “base stacking” phenomenon. Base stacking has been reported to stabilize hybridization and allow efficient capture of dilute nucleic acids by hybridization (38-42). The data generated in Example 6, infra, demonstrates that detection and capture oligonucleotides which bind in tandem result in significantly higher hybridization signals.
Detection Modalities:
The detection zone (detection membrane) of the lateral flow device may comprise one or more capture oligonucleotides which are complementary to one or more target sequences. The capture oligonucleotides are stably affixed to the sample-exposed surface(s) of the microporous detection membrane using standard methodologies. Protein capture reagents may also be patterned onto the detection membrane using standard methods.
The LFM devices of the invention can make use of diverse detection modalities, including visual detection signals resulting from the capture and increased local concentration of an appropriate detection particle. The resulting colorimetric signal can be visualized by eye. Alternatively, for more quantitative and sensitive detection of signal, an electronic instrument capable of detecting colorimetric signals may be employed. Such instruments include standard flatbed scanners, dedicated lateral flow chromatographic strip readers (e.g. QuadScan, KGW Enterprises, Inc), or a simple CCD based devices fabricated for the detection of colorimetric signals such as those employed by commercially available immunochromatographic test strips (e.g. Clearblue Easy Digital Pregnancy Test).
Visualization by eye can be aided by the fabrication of the device in a manner that generates an easily recognized or interpreted shape on the dipstick surface. One example would be the patterning of an LFM with elements in a physical configuration that results in the appearance of a letter or symbol indicative of a positive or negative result (e.g. a “+” or “−” symbol).
Embodiments that employ fluorescent detection reagents such as fluorescent nanoparticles (e.g. Qdots, QuantumDots, Inc.) offer the potential increased sensitivity that results from the application of fluorescence detection technology. Such embodiments can be read using any of a number of ultraviolet light sources including hand held UV lamps, UV emitting LEDs, and light sources with sufficient emission in the UV to excite the nanoparticles. A simple filter can be used to enhance the visualization of nanoparticle fluorescence emissions. For example, a long pass filter with a cut off below the emission wavelength of the nanoparticle may be employed. In the case of excitation with a white light source, an additional filter to limit excitation to UVA and shorter wavelengths can be used (e.g., a 380 nm short pass filter).
The microporous detection membrane may contain capture oligonucleotides printed monolithically in order to produce virtually any colorimetric pattern that can be visualized by the unaided human eye, such as bands, letters, numbers, symbols, and the like. If the sample contains both the first and second target sequences, colored beads with hybridized detection oligonucleotide-target nucleic acid will then hybridize to the immobilized capture oligonucleotide, and thereafter remain stably immobilized to the membrane at that physical location. Such “low density” components of the detection zone may be used to provide a rapid indication of the presence of a target sequence or sequences in the sample, visualized only be the unaided eye.
In addition, the capture zone may contain one or more “high density” components, capable of providing high resolution detail of the signatures of the sequences present in the sample nucleic acid. For example, an array of a number of distinct second detection oligonucleotides may be deposited in distinct physical locations on the membrane (i.e., an array of spots), each of which detection oligonucleotide is specifically complementary to a distinct target sequence. Such high-density arrays may be used to interrogate the sample for genotype signature sequences and the like. These array components may be read by methods well known in the art, including by scanning and computer assisted densitometry, the use of CCD cameras, etc.
Assay devices of the invention comprising such low and high density detection zones are termed “dual-density” systems, assays and devices. The principal design element of such dual-density devices is the provision of two levels of information obtained from a single sample. The low density component provides instantaneous visual information indicative of the presence or absence of a first level target sequence, and may be used to provide fundamental diagnostic information, such as the presence of a nucleic acid sequence indicative of a virus or bacteria in the sample. Because this information is provided by a colored band or other shape or symbol, the user is able to identify the presence of a target immediately and without the use of any instrumentation whatsoever.
The high density components may be assayed using standard instrumentation at any time following the assay. For example, the device may be stored or shipped for high density array analysis using appropriate instrumentation and/or expertise. Thus, as an example, such dual-density devices may be used by a consumer patient for determining whether a body fluid sample contains an influenza virus. A positive result indicates the need for having the high density component of the device analyzed by specialized personnel, in order to determine the influenza strain, subtype, or genotype, for example. The consumer patient is able to use the device to determine the need for profession medical attention. The medical professional is able to analyze the same device for more specific diagnostic information.
LFM Nucleic Acid Assays:
In one aspect of the invention, an LFM assay is provided. LFM assays of the invention are useful for the specific detection of a target analyte, typically from a complex sample of interest, and generally comprise the steps of extracting analyte material (i.e., DNA, RNA, protein) from sample of interest, enriching for the analyte, and detecting the presence of the analyte using an LFM device populated with target-specific capture elements.
In one aspect, the invention provides 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 a lateral flow chromatographic device of the invention, 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.
Various DNA and RNA extraction methodologies are routine and well known in the art. Various kits for the efficient extraction of total nucleic acid, RNA or DNA are widely available from a number of commercial entities. Any of these methodologies and kits may be used to extract nucleic acid from a sample to assessed using the LFM assay.
Single-stranded RNA or DNA targets may be amplified directly, while double-stranded DNA targets generally are rendered single-stranded before amplification. Methods for rendering single-stranded DNA templates from a double-stranded DNA targets include without limitation heat penetration (i.e., 95° C. for 5 minutes) and chemical denaturation (i.e., sodium hydroxide, followed by neutralization). Another method for rendering amplifiable single-stranded DNA from double-stranded DNA involves enzymatic unwinding of the double-stranded DNA, using for example a DNA helicase, which can open-up portions of the DNA, permitting primer and polymerase access and binding (see Kornberg and Baker, 1992, DNA Replication, 2nd edn, New York: WH Freeman and Company; Caruthers and McKay, 2002, Helicase structure and mechanism. Curr Opin Struct Biol 12: 123-133).
As used herein, a “target sequence” is a nucleotide sequence within a target nucleic acid molecule which is to be amplified. Within the target sequence is a primer binding portion, to which primers are designed to hybridize in order to initiate DNA polymerization.
The selection of a particular target sequence for amplification will relate to the LFM assay objectives. For example, where amplification is aimed at identifying a particular strain of an organism, the target sequence should be one of the unique genetic signatures which differentiates that strain from others to which it may be related. In some cases, this may be a single defining sequence. In other cases, a combination of target sequences may be required to reliably identify and differentiate the organism. The selection of target sequences which impart specificity to assays utilizing amplified genetic material involves considerations well known in the art, including for example, unique pathogen-specific sequences, toxins genes, virulence factors or specific signature sequence combinations.
In the practice of the invention, single or multiple target sequences may be amplified in a single reaction using suitable, specific primer oligonucleotides. When multiple target sequences are to be amplified, primers must be designed to avoid possible nonspecific interactions as is well known.
Extracted nucleic acids may be purified prior to amplification. A number of column type DNA and RNA purification devices are commercially available and may be employed for this purpose. Various other techniques for purifying DNA and RNA may be employed, including without limitation, electrophoresis, gradient separation, affinity purification, etc.
LFM assays are useful for the detection of single stranded amplification products derived from samples of interest (i.e., clinical samples, environmental specimens, etc.). LFM is compatible for use with virtually any nucleic acid amplification method. In the context of the rapid, simplified and highly sensitive LFM assays of the invention, LFMs are particularly intended for use with isothermal amplification technologies. In one embodiment, extensively characterized herein by way of the several Examples which follow, the isothermal amplification NASBA is utilized. NASBA-amplified target nucleic acids are detected at very high specificity in a matter of seconds.
NASBA is an RNA amplification methodology that offers several advantages over other RNA amplification methods, including the absence of a reverse transcriptase step. NASBA is an isothermal reaction performed at 41° C., which obviates the need for a thermocycler and may facilitate the production of point-of-test devices. A single-stranded antisense RNA product is produced during NASBA, which can be directly hybridized by a probe sequence to accelerate post-amplification interrogation of the product. Additionally, selection criteria for NASBA primers are less stringent than with other amplification methods, allowing easier primer design in selected less-conserved regions of the gene. Furthermore, the amplification power of NASBA has been reported to be comparable to, or sometimes even higher than that of PCR.
In this connection, the invention provides 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 27.
In the LFM assay progression, initially, and typically following extraction and amplification of target nucleic acid, a solution containing one or more target sequences to be detected by the device is introduced to the sample pad. This may be achieved by dipping the lateral flow device sample pad/sample receiving zone into the solution, or by dropping a quantity of the solution onto the sample pad/sample receiving zone of the lateral flow device. The device is sufficiently robust that the composition of the buffer solution carrying the target sequence(s) is not critical, however, several practical considerations are taken into account to assure compatibility of the buffer with the device. Most significantly, the ionic strength of the sample buffer must be such that precipitation or aggregation of the detection particles does not occur. Similarly, sufficient ionic strength of the buffer is required to support hybridization during lateral flow. Impregnation of the sample pad and/or conjugate release pad with Triton-X100, SDS, BSA, ficol, and/or polyvinyl pyrolidone, or introduction of these components to the sample buffer itself, can stabilize the detection particles and block non-specific interactions between the detection particles and the detection membrane. While a range of concentrations of these reagents can be used successfully, buffers of proven efficacy include 0.1% ficol, 0.1% BSA, 1% Triton X-100, and 150 mM NaCl. This particular buffer supports mono-disperse detection particle suspensions.
Additionally, buffers containing higher concentrations of Triton X-100 and SDS have been found to support higher ionic strength environments without detection particle aggregation and may be used to facilitate hybridization. For example, 3% Triton X-100, 0.1% SDS, 600 mM NaCl has been shown to support subnanomolar hybridization-based detection on the device.
Optimized buffer, reagent parameters and coupling protocols for LFM devices utilizing nitrocellulose detection membranes are presented in Example 5.
Once on the sample pad/sample receiving zone, the analyte solution flows from the proximal (sample) end towards the distal (detection) end of the device. In one embodiment, detection oligonucleotide-functionalized dyed microbeads are embedded into the conjugate release pad component of the device, preferably in lyophilized form, ready to be re-hydrated as the analyte solution travels into this area of the device. As the analyte solution moves across the conjugate release pad, the microbeads are rehydrated and are available for detection oligonucleotide hybridization to target sequences within the sample. Target sequences, when present, will become hybridized to the detection oligonucleotide and thus to the beads. This complex continues lateral flow migration to the detection membrane, where immobilized capture oligonucleotides hybridize to the target sequence, thus capturing the target sequence-bead complex.
The invention also provides lateral flow chromatographic microarray devices. In one aspect, for example, the invention provides 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.
Another aspect is drawn to lateral flow chromatographic microarray devices which eliminate the labeling zone. For example, the invention provides 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.
Kits are also provided. In aspects in which the labeling zone is eliminated, thereby requiring the addition of a labeled detection oligonucleotide, the invention provides a kit for testing the presence of a target nucleic acid in a sample, comprising: (a) a lateral flow chromatographic device or lateral flow chromatographic microarray device of the invention, and (b) a labeled detection oligonucleotide complementary to a second sequence in the target nucleic acid.
LFM, LFM assays and LFM devices of the invention are further described by way of the following examples, none of which are intended to be limiting.
EXAMPLES Example 1 Detection of DNA Hybridization Over Broad Range of Capture Oligonucleotide Deposition Concentrations on DNA DipstickDNA dipstick microarrays were fabricated at a density of 36 features per mm2 using varying concentrations of capture oligonucleotide, as indicated In
The following example demonstrates sensitive detection of single-stranded DNAs using hybridization-based capture and dyed-microsphere colorimetric detection.
Sequences derived from the B. anthracis pagA, capB and cya genes were used to demonstrate multiplexed detection. DNA Dipsticks were patterned with capture sequences for the detection of fragments of three key virulence factors of B. anthracis.
The capture sequences used were:
Populations of beads coupled independently to these detection sequences were pooled for use as colorimetric labels in hybridization-based lateral flow detection assays. The results are shown in
The sensitivity and time required to detect nucleic acids on DNA dipsticks and DNA dipstick microarrays were evaluated using synthetic target molecules for which exact concentrations could be determined.
Dipsticks were fabricated by manual deposition of capture oligonucleotides onto membrane strips of approximately 160 to 275 mm2 surface area, features sizes were ˜2-3 mm in diameter. Dipstick microarrays were printed using microarray fabrication robotics to pattern membrane strips of approximately 60 mm2 surface area such that feature sizes were 300-600 μm in diameter. DNA dipsticks were challenged with 400 μl of synthetic target molecule in the presence of appropriate detection microspheres. Typical lateral flow time for these strips was approximately 45 minutes from sample introduction to complete transport of the sample through the dipstick matrix. Dilution series experiments revealed the sensitivity of detection to be 25 fmol (i.e. 400 μl of 62.5 pM target) (see
To determine the effect of strip surface area and feature diameter on the speed and sensitivity of detection, lateral flow microarray strips were introduced to 10 μl of sample solution containing 20 nM, 2 nM and 0 mM target concentration (
Materials and Methods:
LFM Fabrication: Lateral flow microarrays (LFMs) were printed using a NanoPlotter 2.0 (GeSim, mbH, Dresden, Germany) non-contact picoliter deposition system equipped with NanoTips (GeSim). Unless otherwise indicated, LFMs were patterned with 400 μM solutions of oligonucleotide in H2O containing a 1:50 dilution of Ponceau S (P7767, Sigma) as a tracking dye. A lateral flow compatible nitrocellulose membrane (HiFlow 135, Millipore) was used as the LFM substrate. Following oligonucleotide deposition, nitrocellulose membranes were air dried and exposed to 5000 μJ UV in a StrataLinker (Stratagene). The resulting membrane sheets were cut into 3 mm wide, 30 mm long strips which were either used directly with buffer suspended dyed microspheres or assembled with conjugate release pads into a custom plastic housing. Housings were fabricated from polycarbonate sheet cut using a CO2 laser (VersaLaser VL-300, Universal Laser Systems, Inc., Scottsdale, Ariz., USA). Conjugate release pads were made by impregnating glass fibre conjugate pad (GFCP203000, Millipore) with dyed microspheres covalently conjugated to R-57-76-3TN (see below) in 1% SDS. Microsphere saturated release pads were allowed to air dry under ambient conditions prior to assembly with LFM membranes.
Capture and Detection Oligonucleotides: Table 1 provides capture and detection oligonucleotide sequences, their binding sites within the plcR amplicon are depicted in
Conjugation of Detection Oligonucleotides to Dyed Microspheres: SPHERO™ carboxyl-polystyrene 0.35 μm blue microspheres (Spherotech) were covalently conjugated to amino modified oligonucleotide R-57-76-3TN using the coupling agent 1-etyl-3-(3-dimethylaminopropyl-diimide HCl (EDAC, Pierce) under conditions adapted from Spiro et al (32). Briefly, 4×1010 microspheres were suspended in 100 mM 2-(N-morpholino)ethanesulfonic acid pH 4.5 (MES, Sigma). Indicated amounts of oligonucleotide were introduced to MES suspended microspheres, vortexed and incubated in the presence of 0.5 mg/ml EDAC. Reactions were protected from light in aluminum foil wrapped tubes and incubated at room temperature for 30 min followed by the introduction of additional EDAC to bring the final EDAC concentration to 1 mg/ml. Incubation was continued for an additional 30 min after which beads were washed once with 1 ml 0.02% tween-20 (Sigma) and twice with 0.5 ml 0.1% SDS (Fisher Scientific). Beads were resuspended in 0.5 ml DNAase/RNAase free H2O. Bead suspensions were assessed for aggregation by phase-contrast light microscopy using a Zeiss IM135 inverted microscope.
Results:
Oligonucleotides for hybridization sandwich assays were designed to detect NASBA amplified B. anthracis plcR mRNA or synthetic targets based on relevant subregions of the plcR sequence. Oligonucleotides immobilized on the lateral flow substrate are referred to here as capture oligonucleotides while those conjugated to dyed microspheres for signal generation are referred to as detection oligonucleotides. Supported large pore nitrocellulose membranes were patterned with varying concentrations of capture oligonucleotides using a NanoPlotter™ 2.0 robotic positioning system (GeSiM, Groβerkmannsdorf, Germany) and NanoTip piezoelectronically actuated micropipets (GeSiM). Oligonucleotide dnaR89 was printed on LFM substrates as a positive hybridization control as this oligonucleotide carries sequence complementary to bead coupled detection oligonucleotide R-57-76-3TN. Negative hybridization controls included V24-43 (the reverse complement of capture oligonucleotide R-24-43) and an unrelated sequence complementary to a region of the F. tularensis sdhA locus (FT-S18) (Pardington et al., submitted). By ejecting droplets from the micropipet at a distance of 500 μm from the nitrocellulose substrate, microarray feature sizes of approximately 200 μm could be generated. In contrast to contact microarray printing methods, this approach preserves the fragile pore structure of the membrane required for microsphere-based detection. Patterned nitrocellulose sheets were cut into 3 mm wide strips and then assembled with conjugate release pads in a custom designed plastic housing. An example of the resulting device is shown in
Materials and Methods:
LFM Fabrication: LFMs were fabricated as described in Example 4, infra.
Detection and Capture Oligonucleotides: Detection and capture oligonucleotides were designed as indicated in Example 4, infra. Conjugation of detection oligonucleotides to dyed, polystyrene microspheres was as described in Example 4.
Target Nucleic Acids: A DNA oligonucleotide, dnaR89, composed of sequence derived from a region of the plcR gene of B. anthracis, as shown in
Detection Protocol: Following completion of sample flow, LFM membranes were allowed to air dry prior to scanning with a standard flatbed PC scanner (CanoScan 9950F, Canon, Inc.). Scans were performed at 2400 dpi resolution using 48 bit color. The resulting image files were converted to grayscale, inverted and saved as 16-bit TIFF files using Photoshop CS2 (Adobe). Image files were then analyzed using GenePix Pro 6.0 (Molecular Devices) to quantify microarray spot intensities for NASBA product detection and for dnaR89 dilution series experiments.
Results:
LFMs were fabricated using varying concentrations of capture oligonucleotide to determine optimum printing concentrations. Following lateral flow of 25 fmol dnaR89 in 4×SSC, 5% formamide, 1.4% Triton X-100, 0.1% SDS containing 0.5% R-57-76-3TN coupled microspheres LFMs were scanned on a flatbed scanner and the resulting images quantified. For all capture sequences examined, 400 μM oligonucleotide printing concentrations provided the most favorable signal intensity (
LFM Running Buffer Optimization:
Lateral flow running buffer was based on the widely used standard sodium citrate buffer (SSC) supplemented with 1.4% Triton X-100 and 0.1% SDS to reduce microsphere aggregation and 5% formamide to increase hybridization stringency and destabilize target secondary structure. Given the profound impact ionic strength has on the stringency of DNA hybridization (33,34), SSC concentration was varied from 1× to 9× and assay performance evaluated by densitometry of lateral flow microarrays following hybridization sandwich assays conducted using 25 fmol of the synthetic target dnaR89 or approximately 200 fmol of plcRivt.
To determine the optimum concentration of formamide in LFM running buffer, a series of LFM experiments were conducted at varying formamide concentrations using both dnaR89 and plcRivt. 10 μl of 4×SSC, 1.4% Triton X-100 and 0.1% SDS containing 25 fmol dnaR89 or approximately 200 fmol plcRivt and varying concentrations of formamide, as indicated in
Optimization of Oligonucleotide-Detection Microsphere Coupling:
Given that higher stock concentrations of synthetic oligonucleotide dnaR89 could be obtained which allowed high confidence quantification of this synthetic target relative to what could be achieved with comparatively dilute solutions of the in vitro transcription product plcRivt, subsequent LFM characterization studies made use of dnaR89. The similarity of buffer optima displayed dnaR89 and plcRivt synthetic targets supported the assertion that dnaR89 could be used as an accurate proxy for the performance of LFM assays for NASBA product detection. Others have reported similar findings concluding that appropriately designed DNA oligonucleotides can be used as synthetic targets for the development of assays ultimately used for NASBA product detection (35). Therefore, subsequent LFM assay optimization and characterization was conducted using dnaR89.
To determine the optimum ratios for cross-linking detection oligonucleotides to dyed polystyrene microspheres, we examined populations of beads coupled to oligonucleotide at varying ratios. The 3′ amine modified detection oligonucleotide R-57-76-3TN was covalently linked to polystyrene dyed microspheres using EDAC. The resulting bead/oligonucleotide complexes were evaluated for their ability to mediate detection of dnaR89 in a hybridization sandwich assay. Coupling reactions using a 2.2×104:1 oligonucleotide to bead ratio were found to provide optimum signal as determined by densitometry (
Materials and Methods:
LMF fabrication, oligonucleotide conjugation protocols, target nucleic acids and detection protocols were as described in Examples 4 and 5, supra.
Variable Detection Oligonucleotides: Detection oligonucleotide R-57-76-3TN carrying a 3′ spacer region consisting of 18 T residues was compared to detection oligonucleotide R-57-76-3N, carrying the same analyte complementary sequence as R-57-76-3TN but without the T18 spacer, in order to evaluate whether the poly-T spacer influences accessibility of microsphere-coupled detection oligonucleotides to their hybridization targets.
Variable Capture Oligonucleotides: To determine the relative performance of hybridization sandwich assays making use of capture oligonucleotides with complementarity to different locations of the target sequence, three capture oligonucleotides were synthesized and compared using sandwich assays employing detection oligonucleotide R-57-76-3TN coupled dyed microspheres. R-77-96 was designed to participate in base stacking with R-57-76-3TN when hybridized to the target. Base stacking has been reported to stabilize hybridization and allow efficient capture of dilute nucleic acids by hybridization (38-42). The binding sites for the three capture oligonucleotides examined (R-77-96, R-36-55, and R-24-43) are illustrated in
Results:
Detection oligonucleotide spacer improves hybridization efficiency: The detection oligonucleotide R-57-76-3TN carried a 3′ spacer region consisting of 18 T residues to increase the accessibility of bead bound oligonucleotides for hybridization. R-57-76-3N, which carried the same analyte complementary sequence as R-57-76-3TN but without the T18 spacer, was found to exhibit significantly reduced hybridization to dnaR89 consistent with prior reports that a poly(dT) spacer sequence increases hybridization efficiency to solid-phase coupled oligonucleotides (36,37). T18 spacers were not incorporated into LFM immobilized capture oligonucleotides as they were found to be dispensable for hybridization.
Base stacking effect: Background corrected signal intensity was determined from LFM images using GenePix Pro 6.0 microarray data extraction software. The results, presented in
LFM Detection Sensitivity: To define the detection limit of the LFM assay, a one-tailed t-test was used to determine quantities of dnaR89 that generated signal intensities significantly above 0 amol negative controls. Signals generated at R-77-96 capture features with 250 amol and greater quantities of dnaR89 were significantly higher than 0 amol dnaR89 controls (p<0.05, n=6). By the same criterion, 1 fmol dnaR89 detection limits were obtained for both R-24-43 and R-36-55 (p<0.05, n=6).
Materials and Methods:
LMF fabrication, oligonucleotide conjugation protocols, target nucleic acids and detection protocols were as described in Examples 4 and 5, supra.
For these time course studies, LFM assays were recorded using a digital video recorder (DCR-PC1, Sony). Video frames were collected for quantification using iMovie (Apple Computer). Feature intensity was quantified for time course studies and some optimization experiments using uncalibrated optical density in ImageJ (http://rsb.info.nih.gov/ij/). For better reproduction contrast, LFM images used for figures were cropped and modified by applying the Auto Contrast function in Photoshop CS2. No other modifications were applied.
Results:
The small sample volumes used for LFM detection and the reduced surface area traversed during capillary lateral flow significantly reduces detection times for the LFM relative to traditional lateral flow devices. To quantitatively present the speed of
LFM-mediated nucleic acid detection, we used digital video to follow hybridization sandwich assay mediated detection of synthetic target molecule dnaR89. These studies were conducted over a range of target concentrations using 10 μl of LFM running buffer containing suspended R-57-76-3TN conjugated dyed microspheres. Individual frames were isolated from video data sets and quantified for relative signal intensity over the course of capillary lateral flow across the LFM substrate. The resulting signal data was plotted versus time in seconds as shown in
Materials and Methods:
LMF fabrication, oligonucleotide conjugation protocols, target nucleic acids and detection protocols were as described in Examples 4 and 5, supra. Table 1 provides capture and detection oligonucleotide sequences; binding sites within the plcR amplicon are depicted in
RNA Isolation:
Total RNA was isolated from B. anthracis strain Sterne 7702 and B. thuringiensis strain HD 621 (29) using a previously reported protocol (30). Purified RNA was quantified by measuring OD260 and evaluated by gel electrophoresis. 3×108 cells were used for RNA isolation typically yielding 50-75 μg of total RNA.
Amplification Primer Design
Nucleic acid sequence based amplification (NASBA, (20)) primers, plc-P1 and plc-P2, were designed to amplify a fragment of the plcR locus from B. anthracis. Primer sequences used for NASBA reactions are provided in Table 1, the T7 promoter sequence is italicized in plc-P1. Plc-P1 hybridizes to the plcR transcript such that the 3′ end of the primer forms a base pair with the previously reported polymorphism strictly associated with B. anthracis (27,28). The NASBA P2 primer, plc-P2, is located such that the amplified RNA resulting from NASBA is 179 bases in length, see
Nucleic Acid Sequence-Based Amplification (NASBA)
NASBA reactions were prepared according to the manufacturer's instructions using the NucliSens Basic kit (Biomerieux) and primers plc-P1 and plc-P2 at 0.4 μM each. Amounts of total cellular bacterial RNA were varied, as indicated, between 0 and 2 ng. B. anthracis Sterne 7702 was used as a test strain and B. thuringiensis strain HD 621 was employed as a negative control. 1 μg of human total cellular RNA isolated from HeLa S3 cells (Stratagene) was included in all NASBA reactions to provide a complex RNA background consistent with the composition of human diagnostic samples. Following a 60 minute incubation at 41° C., NASBA reaction products were detected by using a lateral flow microarray (LFM).
Detection of NASBA reaction products: Detection of NASBA products was accomplished by introducing a 2 μl aliquot of a 20 μl NASBA reaction into 8 μl of LFM running buffer (final buffer composition: 4×SSC, 0.1% SDS, 1.4% Triton X-100, 5% deionized formamide, and 0.5% w/v R-57-76-3TN coupled 0.35 μm dyed microspheres). The final volume of solution applied to LFMs was 10 μl. Following completion of sample flow, LFM membranes were allowed to air dry prior to scanning with a standard flatbed PC scanner (CanoScan 9950F, Canon, Inc.). Scans were performed at 2400 dpi resolution using 48 bit color. The resulting image files were converted to grayscale, inverted and saved as 16-bit TIFF files using Photoshop CS2 (Adobe). Image files were then analyzed using GenePix Pro 6.0 (Molecular Devices) to quantify microarray spot intensities for NASBA product detection and for dnaR89 dilution series experiments.
Results:
Prior reports have described a single nucleotide polymorphism (SNP) present in B. anthracis but not close phylogenetic near neighbors including B. cereus and B. thuringiensis (27,28). This SNP has been used as the basis for a sensitive and highly discriminatory real-time PCR assay for B. anthracis (28). To determine the utility of LFM technology for detecting minority nucleic acids in complex samples, NASBA primers were designed to amplify the plcR allele of B. anthracis.
Varying amounts of total cellular RNA isolated from B. anthracis or 2 ng of B. thuringiensis HD 621 RNA as a negative control were introduced to 1 μg of total human cellular RNA isolated from HeLa S3 cells. The resulting mixtures were subjected to NASBA amplification using plc-P1 and plc-P2 primers. Human RNA was included in NASBA amplification reactions to approximate the nucleic acid complexity expected in human diagnostic specimens. 2 μl of NASBA reaction mixture was removed after a 60 minute incubation at 41° C., mixed with 8 μl of LFM running buffer and assayed for plcR amplicon by LFM. Dyed microspheres cross-linked to R-57-76-3TN were used for detection of NASBA amplicons captured on LFMs carrying R-77-96. Data from these studies are presented in
To assess the impact of a fluorescent reporter on the linear dynamic range of LFM mediated analyte detection, a combined colorimetric and fluorescent detection scheme was devised. In this detection scheme, conjugated dyed microspheres as well as streptavidin conjugated fluorescent semi-conductor nanocrystals (605 nm emission, Qdots, InVitrogen, Inc.) are used simultaneously as the reporter particles. For these experiments a detection oligonucleotide, R-57-76-3TBIO (5′-AGGTGAGACATAATCATGCATTTTTTTTTU-biotinTTTTU-biotinTTTTU-biotin3′) [SEQ ID O: 13], carrying three biotin-modified nucleotides was employed in hybridization sandwich assays. Following lateral flow of 250 amol of synthetic analyte dnaR89 in 10 μl of standard LFM running buffer, LFM strips were photographed under ambient light and under illumination with a hand-held UV-LED flashlight (
As illustrated in
To quantify fluorescent nanocrystal LFM signal response linearity, LFMs were challenged with 1, 5, 10, 50, 100, 500, 1000 fmol of dnaR89. Following lateral flow of these samples, LFM strips were adhered to a glass microscope slide and scanned using a standard scanning laser microarray reader (GenePix 4200 Pro, Axon Instruments). A 488 nm laser was used as the excitation source. The resulting data, shown in
-
- 1. Huckle, D. (2006) Point-of-care diagnostics: will the hurdles be overcome this time? Expert Rev Med Devices, 3, 421-426.
- 2. Yang, S. and Rothman, R. E. (2004) PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings. Lancet Infect Dis, 4, 337-348.
- 3. Chin, C. D., Linder, V. and Sia, S. K. (2007) Lab-on-a-chip devices for global health: past studies and future opportunities. Lab Chip, 7, 41-57.
- 4. Koch, W. H. (2004) Technology platforms for pharmacogenomic diagnostic assays. Nat Rev Drug Discov, 3, 749-761.
- 5. Mackay, I. M. (2004) Real-time PCR in the microbiology laboratory. Clin Microbiol Infect, 10, 190-212.
- 6. Cirino, N. M., Musser, K. A. and Egan, C. (2004) Multiplex diagnostic platforms for detection of biothreat agents. Expert Rev Mol Diagn, 4, 841-857.
- 7. Petrik, J. (2006) Diagnostic applications of microarrays. Transfus Med, 16, 233-247.
- 8. Heller, M. J. (2002) DNA microarray technology: devices, systems, and applications. Annu Rev Biomed Eng, 4, 129-153.
- 9. Peytavi, R., Raymond, F. R., Gagne, D., Picard, F. J., Jia, G., Zoval, J., Madou, M., Boissinot, K., Boissinot, M., Bissonnette, L. et al. (2005) Microfluidic device for rapid (<15 min) automated microarray hybridization. Clin Chem, 51, 1836-1844.
- 10. Wei, C. W., Cheng, J. Y., Huang, C. T., Yen, M. H. and Young, T. H. (2005) Using a microfluidic device for 1 microl DNA microarray hybridization in 500 s. Nucleic Acids Res, 33, e78.
- 11. Lim, D. V., Simpson, J. M., Kearns, E. A. and Kramer, M. F. (2005) Current and developing technologies for monitoring agents of bioterrorism and biowarfare. Clin Microbiol Rev, 18, 583-607.
- 12. Glynou, K., loannou, P. C., Christopoulos, T. K. and Syriopoulou, V. (2003) Oligonucleotide-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for DNA analysis by hybridization. Anal Chem, 75, 4155-4160.
- 13. Rule, G. S., Montagna, R. A. and Durst, R. A. (1996) Rapid method for visual identification of specific DNA sequences based on DNA-tagged liposomes. Clin Chem, 42, 1206-1209.
- 14. Dineva, M. A., Candotti, D., Fletcher-Brown, F., Allain, J. P. and Lee, H. (2005) Simultaneous visual detection of multiple viral amplicons by dipstick assay. J Clin Microbiol, 43, 4015-4021.
- 15. Kozwich, D., Johansen, K. A., Landau, K., Roehl, C. A., Woronoff, S. and Roehl, P. A. (2000) Development of a novel, rapid integrated Cryptosporidium parvum detection assay. Appl Environ Microbiol, 66, 2711-2717.
- 16. Zuiderwijk, M., Tanke, H. J., Sam Niedbala, R. and Corstjens, P. L. (2003) An amplification-free hybridization-based DNA assay to detect Streptococcus pneumoniae utilizing the up-converting phosphor technology. Clin Biochem, 36, 401-403.
- 17. Zijlmans, H. J., Bonnet, J., Burton, J., Kardos, K., Vail, T., Niedbala, R. S. and Tanke, H. J. (1999) Detection of cell and tissue surface antigens using up-converting phosphors: a new reporter technology. Anal Biochem, 267, 30-36.
- 18. Duck, P., Alvarado-Urbina, G., Burdick, B. and Collier, B. (1990) Probe amplifier system based on chimeric cycling oligonucleotides. Biotechniques, 9, 142-148.
- 19. Piepenburg, O., Williams, C. H., Stemple, D. L. and Armes, N. A. (2006) DNA detection using recombination proteins. PLoS Biol, 4, e204.
- 20. Compton, J. (1991) Nucleic acid sequence-based amplification. Nature, 350, 91-92.
- 21. Kievits, T., van Gemen, B., van Strijp, D., Schukkink, R., Dircks, M., Adriaanse, H., Malek, L., Sooknanan, R. and Lens, P. (1991) NASBA isothermal enzymatic in vitro nucleic acid amplification optimized for the diagnosis of HIV-1 infection. J Virol Methods, 35, 273-286.
- 22. Malek, L., Sooknanan, R. and Compton, J. (1994) Nucleic acid sequence-based amplification (NASBA). Methods Mol Biol, 28, 253-260.
- 23. Fong, W. K., Modrusan, Z., McNevin, J. P., Marostenmaki, J., Zin, B. and Bekkaoui, F. (2000) Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology. J Clin Microbiol, 38, 2525-2529.
- 24. Baeumner, A. J., Schlesinger, N. A., Slutzki, N. S., Romano, J., Lee, E. M. and Montagna, R. A. (2002) Biosensor for dengue virus detection: sensitive, rapid, and serotype specific. Anal Chem, 74, 1442-1448.
- 25. Baeumner, A. J., Pretz, J. and Fang, S. (2004) A universal nucleic acid sequence biosensor with nanomolar detection limits. Anal Chem, 76, 888-894.
- 26. Hartley, H. A. and Baeumner, A. J. (2003) Biosensor for the specific detection of a single viable B. anthracis spore. Anal Bioanal Chem, 376, 319-327.
- 27. Zaytseva, N. V., Montagna, R. A., Lee, E. M. and Baeumner, A. J. (2004) Multi-analyte single-membrane biosensor for the serotype-specific detection of Dengue virus. Anal Bioanal Chem, 380, 46-53.
- 28. Edwards, K. A. and Baeumner, A. J. (2006) Optimization of DNA-tagged dye-encapsulating liposomes for lateral-flow assays based on sandwich hybridization. Anal Bioanal Chem, 386, 1335-1343.
- 29. Easterday, W. R., Van Ert, M. N., Simonson, T. S., Wagner, D. M., Kenefic, L. J., Allender, C. J. and Keim, P. (2005) Use of single nucleotide polymorphisms in the plcR gene for specific identification of Bacillus anthracis. J Clin Microbiol, 43, 1995-1997.
- 30. Easterday, W. R., Van Ert, M. N., Zanecki, S. and Keim, P. (2005) Specific detection of bacillus anthracis using a TaqMan mismatch amplification mutation assay. Biotechniques, 38, 731-735.
- 31. Hill, K. K., Ticknor, L. O., Okinaka, R. T., Asay, M., Blair, H., Bliss, K. A., Laker, M., Pardington, P. E., Richardson, A. P., Tonks, M. et al. (2004) Fluorescent amplified fragment length polymorphism analysis of Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis isolates. Appl Environ Microbiol, 70, 1068-1080.
- 32. Pannucci, J., Cai, H., Pardington, P. E., Williams, E., Okinaka, R. T., Kuske, C. R. and Cary, R. B. (2004) Virulence signatures: microarray-based approaches to discovery and analysis. Biosens Bioelectron, 20, 706-718.
- 33. Deiman, B., van Aarle, P. and Sillekens, P. (2002) Characteristics and applications of nucleic acid sequence-based amplification (NASBA). Mol Biotechnol, 20, 163-179.
- 34. Spiro, A., Lowe, M. and Brown, D. (2000) A bead-based method for multiplexed identification and quantitation of DNA sequences using flow cytometry. Appl Environ Microbiol, 66, 4258-4265.
- 35. Albretsen, C., Haukanes, B. I., Aasland, R. and Kleppe, K. (1988) Optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene DNA probes. Anal Biochem, 170, 193-202.
- 36. Schildkraut, C. (1965) Dependence of the melting temperature of DNA on salt concentration. Biopolymers, 3, 195-208.
- 37. Blake, R. D. and Delcourt, S. G. (1996) Thermodynamic effects of formamide on DNA stability. Nucleic Acids Res, 24, 2095-2103.
- 38. Baeumner, A. J., Leonard, B., McElwee, J. and Montagna, R. A. (2004) A rapid biosensor for viable B. anthracis spores. Anal Bioanal Chem, 380, 15-23.
- 39. Guo, Z., Guilfoyle, R. A., Thiel, A. J., Wang, R. and Smith, L. M. (1994) Direct fluorescence analysis of genetic polymorphisms by hybridization with oligonucleotide arrays on glass supports. Nucleic Acids Res, 22, 5456-5465.
- 40. Day, P. J. R., Flora, P. S., Fox, J. E. and Walker, M. R. (1991.) Immobilization of polynucleotides on magnetic particles: Factors Influencing hybridization efficiency. Biochem. J., 278, 735-740.
- 41. O'Meara, D., Nilsson, P., Nygren, P. A., Uhlen, M. and Lundeberg, J. (1998) Capture of single-stranded DNA assisted by oligonucleotide modules. Anal Biochem, 255, 195-203.
- 42. Lane, M. J., Paner, T., Kashin, I., Faldasz, B. D., Li, B., Gallo, F. J. and Benight, A. S. (1997) The thermodynamic advantage of DNA oligonucleotide ‘stacking hybridization’ reactions: energetics of a DNA nick. Nucleic Acids Res, 25, 611-617.
- 43. O'Meara, D., Yun, Z., Sonnerborg, A. and Lundeberg, J. (1998) Cooperative oligonucleotides mediating direct capture of hepatitis C virus RNA from serum. J Clin Microbiol, 36, 2454-2459.
- 44. Kandimalla, E. R., Manning, A., Lathan, C., Byrn, R. A. and Agrawal, S. (1995) Design, biochemical, biophysical and biological properties of cooperative antisense oligonucleotides. Nucleic Acids Res, 23, 3578-3584.
- 45. Kieleczawa, J., Dunn, J. J. and Studier, F. W. (1992) DNA sequencing by primer walking with strings of contiguous hexamers. Science, 258, 1787-1791.
- 46. Cheek, B. J., Steel, A. B., Torres, M. P., Yu, Y. Y. and Yang, H. (2001) Chemiluminescence detection for hybridization assays on the flow-thru chip, a three-dimensional microchannel biochip. Anal Chem, 73, 5777-5783.
- 47. Roper, M. G., Easley, C. J. and Landers, J. P. (2005) Advances in polymerase chain reaction on microfluidic chips. Anal Chem, 77, 3887-3893.
- 48. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B. and Erlich, H. A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science, 239, 487-491.
All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention, and any which are functionally equivalent are within the scope of the invention. Various modifications to the models and methods of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and teachings, and are similarly intended to fall within the scope of the invention. Such modifications or other embodiments can be practiced without departing from the true scope and spirit of the invention.
Claims
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.
Type: Application
Filed: Feb 1, 2016
Publication Date: Aug 4, 2016
Inventor: Robert B. Cary (Santa Fe, NM)
Application Number: 15/012,804
