EP1848823A2 - Methods for separating short single-stranded nucleic acid from long single- and double-stranded nucleic acid, and associated biomolecular assays - Google Patents

Methods for separating short single-stranded nucleic acid from long single- and double-stranded nucleic acid, and associated biomolecular assays

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Publication number
EP1848823A2
EP1848823A2 EP06719184A EP06719184A EP1848823A2 EP 1848823 A2 EP1848823 A2 EP 1848823A2 EP 06719184 A EP06719184 A EP 06719184A EP 06719184 A EP06719184 A EP 06719184A EP 1848823 A2 EP1848823 A2 EP 1848823A2
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EP
European Patent Office
Prior art keywords
nucleic acid
target
dna
solution
negatively charged
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EP06719184A
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German (de)
French (fr)
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EP1848823A4 (en
Inventor
Lewis J. Rothberg
Huixiang Li
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University of Rochester
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University of Rochester
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Publication of EP1848823A2 publication Critical patent/EP1848823A2/en
Publication of EP1848823A4 publication Critical patent/EP1848823A4/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6832—Enhancement of hybridisation reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the present invention relates to hybridization-based nucleic acid detection procedures and materials for practicing the same.
  • PCR polymerase chain reaction
  • a first aspect of the present invention relates to a method for detecting presence or absence of a target nucleic acid molecule in a test solution (e.g., sample).
  • This method includes the steps of: combining at least one single-stranded oligonucleotide probe with a test solution potentially including a target nucleic acid to form a hybridization solution, wherein the at least one single-stranded oligonucleotide probe and the test solution are combined under conditions effective to allow formation of a hybridization complex between the at least one single-stranded oligonucleotide probe and any target nucleic acid present in the test solution; exposing the hybridization solution to a plurality of metal nanoparticles under conditions effective to allow the at least one single-stranded oligonucleotide probe that remains unhybridized after said combining to associate electrostatically with the plurality of metal nanoparticles; and determining whether the at least one single-stranded oligonucleotide probe has
  • a colorimetric assay utilizes an unlabeled oligonucleotide probe and involves making the determination by detecting a color change of the hybridization solution after the step of exposing, whereby a color change indicates substantial aggregation of the plurality of metal nanoparticles in the presence of the target nucleic acid. If no color change (or an insignificant change) occurs, absence of the target nucleic acid is indicated.
  • Another embodiment utilizes a fluorescently labeled oligonucleotide probe and involves determining whether or not fluorescence can be detected following exposure to the plurality of metal nanoparticles, whereby elimination of fluorescence indicates absence of a target nucleic acid and remaining fluorescence indicates its presence. If fluorescence by the labeled oligonucleotide probes remains, the oligonucleotide probes have formed duplexes and remain dissociated from the metal nanoparticles (i.e., no fluorescence quenching has occurred).
  • a second aspect of the present invention relates to a method for detecting a single nucleotide polymorphism ("SNP") in a target nucleic acid molecule.
  • SNP single nucleotide polymorphism
  • This method is carried out by combining (i) a test solution including a target nucleic acid molecule and (ii) at least one first single-stranded oligonucleotide probe that has a nucleotide sequence that hybridizes to a region of the target nucleic acid molecule that may contain a single-nucleotide polymorphism, to form a test hybridization solution, wherein said combining is carried out under conditions effective to allow hybridization between the target nucleic acid molecule and the at least one first single-stranded oligonucleotide probe to form at least one hybridization complex; combining (i) a control solution including the target nucleic acid molecule and (ii) at least one second single-stranded oligonucleotide probe that has a nucleotide sequence that hybridizes perfectly to a region of the target nucleic acid molecule that does not contain a single-nucleotide polymorphism, to form a control hybridization solution, wherein
  • a third aspect of the present invention relates to a method for detecting a SNP in a target nucleic acid molecule. This method is carried out by: combining (i) a solution including a target nucleic acid molecule and (ii) at least one first single- stranded oligonucleotide probe having a nucleotide sequence and a fluorescent label attached thereto, wherein the nucleotide sequence hybridizes to a region of the target nucleic acid molecule that may contain a single-nucleotide polymorphism, to form a hybridization solution, wherein said combining is carried out under conditions effective to allow hybridization between the target nucleic acid molecule and the at least one first single-stranded oligonucleotide probe to form at least one hybridization complex; exposing the hybridization solution to a plurality of metal nanoparticles under conditions effective to allow unhybridized probes in the hybridization solution to electrostatically associate with the metal nanoparticles;
  • a fourth aspect of the present invention relates to a method for detecting a target nucleic acid in a test solution.
  • This method includes the steps of: subjecting a portion of a test solution potentially including a target nucleic acid to polymerase chain reaction and obtaining a product solution that includes single- stranded nucleic acid products of the polymerase chain reaction; combining at least one single-stranded oligonucleotide probe with the product solution to form a hybridization solution under conditions effective to allow formation of a hybridization complex between the at least one single-stranded oligonucleotide probe and any target nucleic acid present in the product solution; exposing the hybridization solution to a plurality of metal nanoparticles under conditions effective to allow any single- stranded nucleic acids in the hybridization solution to associate with the plurality of metal nanoparticles; and determining whether the at least one single-stranded oligonucleotide probe has hybridized to target nucleic acid or electrostatically associated with
  • a fifth aspect of the present invention relates to a method of detecting a pathogen in a sample that includes the steps of obtaining a sample that may contain nucleic acid of a pathogen, and then performing a method of the present invention using an oligonucleotide probe specific for a target nucleic acid of the pathogen, wherein the step of determining that the at least one single-stranded oligonucleotide probe has hybridized to the target nucleic acid indicates presence of the pathogen.
  • a sixth aspect of the present invention relates to a method of genetic screening.
  • a seventh aspect of the present invention relates to a method of detecting a protein in a sample.
  • This method is carried out by obtaining a sample, performing an immuno-polymerase chain reaction procedure using the sample, wherein the immuno-polymerase chain reaction procedure results in amplification of a nucleic acid that is conjugated to a protein, and then performing a method of the present invention using an oligonucleotide probe specific for the nucleic acid that is conjugated to the protein (or its complement), wherein the step of determining that the at least one single-stranded oligonucleotide probe has hybridized to the target nucleic acid indicates that the protein is present in the sample.
  • An eighth aspect of the present invention relates to a method of quantifying the amount of amplified nucleic acid prepared by polymerase chain reaction.
  • This method is carried out by providing two or more fluorescently labeled oligonucleotide primers that each have a nucleotide sequence capable of hybridizing to a nucleic acid molecule, or its complement, to be amplified; performing polymerase chain reaction using a target nucleic acid molecule and/or its complement, and the provided fluorescently labeled oligonucleotide primers; and performing the fluorimetric method of the present invention on a sample obtained after said performing polymerase chain reaction, wherein the level of fluorescence detected from the sample indicates the amount of primer that has been incorporated into an amplified nucleic acid molecule.
  • a ninth aspect of the present invention relates to a method for detecting presence or absence of a target nucleic acid in a test solution that includes the steps of: combining at least one single-stranded oligonucleotide probe with a test solution potentially including a target nucleic acid to form a hybridization solution, wherein the at least one single-stranded oligonucleotide probe and the test solution are combined under conditions effective to allow formation of a hybridization complex between the at least one single-stranded oligonucleotide probe and any target nucleic acid present in the test solution; exposing the hybridization solution to a plurality of negatively charged nanoparticles under conditions effective to allow any single- stranded oligonucleotide probe or non-target nucleic acid that remains unhybridized after said combining to associate electrostatically with the plurality of negatively charged nanoparticles; separating the plurality of negatively charged nanoparticles from the hybridization solution after said exposing; and determining whether
  • kits containing various components that will allow a user to perform one or more methods of the present invention minimally include a first container that contains a plurality of negatively charged nanoparticles; and a second container that contains a salt solution having a concentration of salt that is effective to cause aggregation of the negatively charged nanoparticles.
  • the kits can further include a third container that contains at least one single-stranded oligonucleotide probe complementary to a target nucleic acid and/or a fourth container that contains a hybridization solution and/or a filter sufficient to allow for filtration of aggregated nanoparticles.
  • the kits can include a container that contains the plurality of negatively charged nanoparticles coupled to a substrate.
  • An eleventh aspect of the present invention relates to a detection device for performing a method of the present invention.
  • Assays and kits of the present invention involve the use of negatively charged nanoparticles and nucleic acid molecules, harnessing the electrostatic interactions between the nanoparticles and nucleic acid molecules.
  • applicants have identified four unique interactions that can be harnessed by the assays and materials of the present invention.
  • the negatively charged nanoparticles in solution are typically stabilized by their repulsion, which prevents the strong Van der Waals attraction between the particles from causing them to aggregate (Hunter, Foundations of Colloid Science, Oxford University Press Inc., New York (2001); Shaw, Colloid and Surface Chemistry, Butterworth-Heinemann Ltd., Oxford (1991), each of which is hereby incorporated by reference in its entirety). Repulsion between the charged phosphate backbone of ds-nucleic acid and the negatively charged nanoparticles dominates the electrostatic interaction between the nanoparticle and ds-nucleic acid so that ds-nucleic acid will not adsorb.
  • the ss-nucleic acid is sufficiently flexible to partially uncoil its bases, they can be exposed to the negatively charged nanoparticles. Under these conditions, the negative charge on the backbone is sufficiently distant so that attractive Van der Waals forces between the bases and the nanoparticle are sufficient to cause ss-nucleic acid to adsorb to the negatively charged nanoparticle. The same mechanism is not operative with ds-nucleic acid because the duplex structure does not permit the uncoiling needed to expose the bases.
  • the selective adsorption of ss-DNA and RNA to negatively charged nanoparticles e.g., citrate-coated Au nanoparticles
  • the assay is easy to implement for visual detection at the level of 100 femtomoles, and it is shown that it is easily adapted to detect single base mismatches between probe and target. Also presented herein are initial studies of how length mismatches between target and probe sequence affect the propensity for oligonucleotides to adsorb on metal nanoparticles. [0023] By harnessing the above-identified interactions in the assays and kits of the present invention, the present invention affords methods of detecting target nucleic acids that offer a number of benefits over previously developed detection procedures.
  • Some of these benefits include: no target labeling is required; the assays occur in solution, allowing for detection of the target nucleic acid in less than about 10 minutes (which is significantly faster than chip or surface-based assays that tend to slow down the hybridization process); the detection procedure is temporally separated from the hybridization procedure so that the hybridization process can be optimized with little or no regard to the detection procedure; and the assays can be performed using commercially available materials.
  • the two basic embodiments of the present invention, a colorimetric assay and a fluorimetric assay afford significant benefits.
  • the colorimetric assay can be performed without the need for expensive detection instrumentation, such as fluorescence microscopes or photomultipliers.
  • Detection of a positive or negative result in the colorimetric assay can be assessed by naked eye of an observer.
  • the assays are extremely sensitive, capable of detecting target nucleic acids in femtomole quantities (or less in the case of the fluorescent approach), capable of discriminating between complex mixtures of nucleic acid, and capable of discriminating between wild-type targets and those bearing SNPs or other mutations such as deletions or modifications such as knockout insertions.
  • Figure 1 is a pictorial representation of the colorimetric method for differentiating between single and double stranded oligonucleotides; and consequently selective oligonucleotide detection.
  • the circles represent colloidal metal (e.g. gold) nanoparticles.
  • Figure 2 is a pictorial representation of the fluorimetric method for selective oligonucleotide detection.
  • the red stars in panels A, B, and D represent identifiable (i.e., unquenched) fluorescence from the fluorescence label on the probe strands.
  • the thin green strands and the thick green strands represent single-strand and double-strand nucleic acid molecules, respectively.
  • the circles in panels C and D represent metal (e.g., gold) nanoparticles. Hybridization between the oligonucleotide probes and target nucleic acid molecules occur before introducing metal nanoparticles.
  • Figure 3 is a schematic protocol of protein detection combining immuno-PCR with the methods of the present invention.
  • Figures 4A-B provide evidence for preferential adsorption of ss-DNA on gold nanoparticles.
  • Figure 4A is a graphical illustration of fluorescence emitted from rhodamine red attached to ss-DNA (dashed) and ds-DNA (solid). The fluorescence spectra were recorded from mixtures consisting of the trial hybridization solution (final concentration of the dye labeled ss-DNA: 50 nM), 500 ⁇ L of gold colloid, and 500 ⁇ L of 10 mM phosphate buffer solution (PBS) containing 0.1 M NaCl. The ss-DNA (dashed) curve was recorded from the mixture containing the probe and its non-complementary target (nc-target). Dot curve was recorded from the W
  • FIG. 4B is a graphical illustration of Surface Enhanced Resonant Raman Scattering ("SERRS") from Rhodamine Green tagged on ss-DNA (solid) and ds-DNA (dashed).
  • SERRS Surface Enhanced Resonant Raman Scattering
  • FIG. 5A-C show colorimetric detection of oligonucleotide hybridization.
  • Figure 5A is a graph showing absorption spectra of gold colloid (diamonds) and the mixtures containing ss-DNA 1 (circles), ss-DNA2 (triangles), and ds-DNA from the hybridization of ss-DNA 1 and ss-DNA2 (squares), respectively.
  • the gold colloid was diluted with water to the same concentration as in the mixtures.
  • the mixtures contained trial hybridization solution (5 ⁇ L (60 ⁇ M) ss-DNA in salt buffer solution) added to 500 ⁇ L of 17 nM gold colloid, followed by 200 ⁇ L of 10 mM PBS and 0.2 M NaCl).
  • Figure 5B is a graphical illustration of the ratio of the absorbance at 520 nm to the absorbance at 700 nm versus oligonucleotide concentration expressed in number of DNA per gold nanoparticle.
  • the DNA sequences and the mixture are the same as in Figure 5 A, except for variation of the amount of DNA.
  • Figure 5C is a photograph showing colorimetric detection of a DNA sequence fragment characteristic of Severe Acute Respiratory Syndrome (“SARS") virus (Drosten et al., The New England Journal of Medicine 348:1967-1976 (2003), which is hereby incorporated by reference in its entirety). All solutions contained 120 picomoles of probe, 200 ⁇ L gold colloid, and 100 ⁇ L of 10 mM PBS and 0.2 M NaCl. The ratio of the amount of target to the amount of probe in the solutions was 0, 0.2, 0.4, 0.6, and 1 (from left to right), respectively.
  • SARS Severe Acute Respiratory Syndrome
  • Figures 6A-E show colorimetric detection of targets in mixtures, low concentrations, low amounts, and with single base mismatches.
  • Figure 6A is a photograph showing detection of a target sequence in a mixture.
  • 3.5 ⁇ L of trial hybridization solution was mixed with 300 ⁇ L of gold colloid and 300 ⁇ L of 10 mM phosphate buffer solution containing 0.2 M NaCl.
  • the complementary target contained in the solutions from left to right were 50%, 40%, 30%, and 0% of the total ⁇
  • FIG. 6B is a photograph showing detection of target DNA in low concentration solution. 100 ⁇ L of gold colloid was diluted in 300 ⁇ L water, mixed with 1 ⁇ L trial hybridization solution and 300 ⁇ L of 10 mM phosphate buffer solution containing 0.3 M NaCl (final target concentration: 4.3 nM). The vial on the left contained unmatched ss-DNA strands while the vial on the right contained complementary strands.
  • Figure 6C is a photograph showing detection of small amounts of target.
  • oligonucleotide and 300 ⁇ L of gold nanoparticles were ultrasonicated for 0.5, 1, and 2 minutes, respectively, and then mixed with 300 ⁇ L of 10 mM phosphate buffer solution 0.3 M NaCl (final target concentration: 0.05 ⁇ M).
  • the solution in the left vial of each dehybridization time group contained ds-DNA with a single base pair mismatch while the right vial contained perfectly matched target and probe strands. The red color indicates that part of ds-DNA has dehybridized.
  • the oligonucleotide sequences are identified in the text.
  • Figures 7A-B show that gold nanoparticles preferentially quench the fluorescence from fluorophores labeled on ss-DNA.
  • Figure 7A is a graph showing the fluorescence spectra of the mixtures of 5 ⁇ L (10 ⁇ M) trial hybridized solution of rhodamine red labeled ss-DNA probe and its complementary target (solid squares), or non-complementary target (open squares), 500 ⁇ L of gold colloid and 500 ⁇ L of 10 mM PBS containing 0.1 M NaCl.
  • Figure 7B is a graph showing the fluorescence image intensity profile measured with a confocal fluorescence microscope.
  • 0.5 ⁇ L (0.1 ⁇ M) of the trial hybridization solution was mixed with 500 ⁇ L of the diluted gold colloid (diluted with deionized water by factor 20) and 500 ⁇ L of 10 mM PBS containing 0.1 M NaCl. Solid circles were recorded from 2 ⁇ L of the mixture containing complementary target; open circles from 2 ⁇ L of the mixture containing non-complementary target.
  • Figures 8A-B show detection of long target and long target in a mixture.
  • Figure 8A is a graph showing the method working with long target. The fluorescence spectra were recorded from the solutions containing complementary target a (solid squares), complementary target b (open squares), and non- complementary target c (solid triangles), respectively. The solution contained 4 ⁇ L (10 ⁇ M) of trial hybridized solution, 500 ⁇ L gold colloid, and 500 ⁇ L of 10 mM PBS containing 0. IM NaCl.
  • Figure 8B is a graph showing the method working with long target in a mixture.
  • the fluorescence spectra were recorded from mixtures containing 1% complementary target a (solid squares), 1% complementary target b (open squares), and non-complementary target (solid triangles), respectively.
  • the components of oligonucleotides in the trial hybridized solution contained 10 picomolar non-complementary target, 0.5 picomolar probe, and 0.1 picomolar candidates.
  • the mixtures were made up of 0.5 ⁇ L of trial hybridized solutions, 500 ⁇ L gold colloid (diluted with 250 ⁇ L water), and 500 ⁇ L of 10 mM PBS containing 0. IM NaCl.
  • Figures 9A-B show single base-pair mismatch detection.
  • Figure 9 A is a graph showing the probe binding in the middle of long target a and target a'.
  • Figure 9B is a graph showing the probe binding at one end of long target b and complementary target b'.
  • the fluorescence spectra for single base-pair mismatch detection were recorded from mixtures containing 1 ⁇ L (10 ⁇ M) trial hybridized solution (same amount of the probe and the target) warmed in 46°C water bath, 500 ⁇ L gold colloid, and 500 ⁇ L of 10 mM PBS and 0.1 M NaCl. Solid squares were recorded from the mixtures containing perfect matched ds-DNA and open squares from the mixtures containing ds-DNA with one base-pair mismatch.
  • Figures 10A-B show simultaneous multiple target detection. Figure
  • FIG. 1OA is a graph showing excitation at 570 nm, which is absorption maximum of rhodamine red tagged on probe 1.
  • Figure 1OB is a graph showing excitation at 648 nM, which is absorption maximum of cy5 tagged on probe 2.
  • the second peak of the spectrum (solid squares) in Figure 1OB is the emission of cy5 tagged on probe 2 excited by 570 nm.
  • Figures 1 IA-D show adsorption of ss-DNA to gold nanoparticles.
  • Figure 1 IA graphically illustrates absorption spectra of 300 ⁇ L gold colloid and 100 ⁇ L deionized water (red), 100 ⁇ L of 10 mM PBS (0.2 M NaCl) (blue), 300 picomoles 24 base ss-DNA first, then 100 ⁇ L of 10 mM PBS (0.2 M NaCl) (green).
  • Figure 1 IB is a graph showing photoluminescence intensity versus time following addition of 4 picomoles rhodamine red tagged ss-DNAs to 1000 ⁇ L gold colloid. 10 mer (red), 24 mer (green) and 50 mer (blue).
  • Figure 11C graphically illustrates absorption spectra of the mixture of 200 picomoles ss-DNA (50 mer) and 300 ⁇ L gold nanoparticles heated at different temperature for two minutes, followed by addition of 300 ⁇ L of 10 mM PBS (0.2 M NaCl). 22 0 C (blue), 45°C (cyan), 70 0 C (green), and 95 0 C (red).
  • Figure 1 ID graphically illustrates the fluorescence spectra of the hybridized solutions of rhodamine red labeled 15 mer ss-DNA, 50 mer ss-DNA, and gold colloid, the 15 mer binding to 50 mer at middle (red), at end (green) and nowhere (blue).
  • Figure 12 is a schematic of the interaction between negatively charged metal nanoparticules and ss-DNA.
  • the wedge-like structure (left) represents the metal nanoparticle, and the structure (right) represents a ss-nucleic acid having a phosphate backbone (solid vertical line) and nucleotide bases (horizontal lines).
  • Figures 13A-B show identification of PCR amplified DNA sequences.
  • Figure 13A is a schematic of the detection protocol.
  • the mixture of PCR product and probes is denatured and annealed below the melting temperature of the complementary probes, followed by addition of gold colloid.
  • the long blue and green lines represent the PCR amplified DNA fragments and the pink and light blue medium bars the excess PCR primers.
  • the short blue and green bars are complementary probes that bind, resulting in gold nanoparticle aggregation (purple color).
  • the short purple and orange bars are non-complementary probes that do not bind and adsorb to the gold nanoparticles, preventing nanoparticle aggregation and leaving the solution pink.
  • Figure 13B is a color photograph of the resulting solutions with complementary probes (a) and non-complementary probes (b).
  • Figures 14A-B show single base-pair mismatch detection.
  • Figure 14A illustrates the detection strategy.
  • the red spots on long green and blue lines represent positions of a potential SNP.
  • the long green and blue lines are the complementary sequences of PCR amplified DNA fragment.
  • the short green and blue bars are probes complementary to parts of the wild type sequence of PCR amplified DNA fragment as illustrated.
  • Figure 14B is a photograph showing detection of a single base-pair mismatch.
  • Vials b, d, and f contain PCR product with probes overlapping the single-base mismatch while vials a, c, and e contain PCR product with probes not overlapping the single base pair mismatch. Photographs were taken of the mixtures annealed at 50 0 C (a, b), 54°C (c, d) and 58°C (e, f). 8 ⁇ L PCR product, 3.5 picomoles probe and 70 ⁇ L gold colloid were used in each vial. [0038] Figures 15 A-B illustrate single base-pair mismatch detection using RNA probes and RNA targets.
  • Figure 16 illustrates schematically one implementation of the immobilized bead method for separating double stranded from single stranded nucleic acids. Removal of unhybridized short ss-DNA probes by processing the analyte through a filter of packed glass beads (circles filled with grid) functionalized with immobilized negatively charged nanoparticles (shaded circles). The trial hybridization solution prior to the filter is shown schematically above and after the filter below.
  • FIG. 17 is a graph showing that ss-DNA is preferentially retained by the column of immobilized beads.
  • Figure 18 is a graph illustrating the fluorescence of solutions remaining after removal of gold by salt-induced crashout and centrifugation.
  • FIGS 19A-D illustrate the colorimetric method for RNA sequence detection.
  • the same mixtures of trial hybridization solutions and gold colloid were used.
  • the left vial in each image contains complementary target
  • the middle vial contains a target with a single base mismatch with the probe
  • the right vial contains a random non-complementary target.
  • Figures 20A-B are graphs showing the absorption spectra from the mixtures of trial hybridization solutions annealed at two different temperatures after being added to gold colloid.
  • Squares, circles and triangles from the mixtures contain, respectively, complementary target (c-target), mismatch target (mc-target) and non- complementary target (nc-target).
  • c-target complementary target
  • mc-target mismatch target
  • nc-target non- complementary target
  • FIG. 21 is a graph illustrating detection of single base mutations in RNA sequences using fluorescence quenching of fluorescently labeled DNA probe. Fluorescence spectra of the mixtures of hybridization solution, gold colloid, and buffer/salt solution are illustrated two minutes after mixing.
  • Figure 22 is a graph illustrating the detection of single base mutations in RNA sequences in complex mixtures using the fluorescence assay.
  • P and T denote probe and target, respectively, while w and m indicate wild-type and mutant, respectively.
  • AU hybridization solutions contain non-complementary background RNA at 10 times the concentration of the target. Sequences of the wild-type probe, wild-type target, and mutant target are stated in the description of Figure 21.
  • the methods of the present invention can be used to detect the presence (or substantial absence) of a target nucleic acid molecule in a sample or test solution.
  • the method involves combining at least one single-stranded oligonucleotide probe and the test solution under conditions effective to allow formation of a hybridization complex between the at least one single-stranded oligonucleotide probe and any target nucleic acid present in the test solution. If no target nucleic acid or substantially no target nucleic acid is present, then no hybridization complex or substantially no hybridization complex will form.
  • the hybridization solution After allowing for hybridization to occur (i.e., if hybridization between the probe and target is possible), the hybridization solution is exposed to a plurality of negatively charged nanoparticles under conditions effective to allow any unhybridized probe to associate electrostatically with the plurality of negatively charged nanoparticles. A determination is then made whether the at least one single-stranded oligonucleotide probe has hybridized to target nucleic acid or electrostatically associated with one or more of the plurality of negatively charged nanoparticles. This determination is made according to an optical property of the hybridization solution, as discussed below.
  • the methods of the present invention can further include a step for separating ds-nucleic acid from the short single-stranded probe molecules (or other ss- nucleic acids) that remain unbound after the hybridization step, as discussed below.
  • the target nucleic acid molecule that is intended to be detected can be
  • DNA or RNA can be isolated directly from samples (i.e., concentrated to be free of cellular debris) and then tested, if present in sufficient quantities, or it can first be amplified by polymerase chain reaction ("PCR") or reverse-transcription PCR.
  • PCR polymerase chain reaction
  • reverse-transcription PCR reverse-transcription PCR
  • the cDNA can also have incorporated therein synthetic, natural, or structurally modified nucleoside bases.
  • the target nucleic acid molecule can also be from any source organism
  • the target nucleic acid can contain a nucleotide sequence coupled or otherwise conjugated to a protein or polypeptide. In such case, detection of the target nucleic acid directly confirms presence of the protein or polypeptide.
  • the target nucleic acid can contain a nucleotide sequence coupled or otherwise conjugated to a protein or polypeptide that participates in an immuno-PCR procedure; the subsequently amplified target cDNA confirms indirectly the presence of the target nucleic acid in a sample to be tested (i.e., absence of the target cDNA confirms that the target is not present in the initial sample).
  • the single-stranded oligonucleotide probes that can be used in the present invention can either be unlabeled or they can be conjugated or otherwise coupled to a label.
  • Suitable labels include, without limitation, fluorescent labels, redox (electrochemical) labels, and radioactive labels.
  • Coupling of a fluorescent label to the oligonucleotide probe can be achieved using known nucleic acid-binding chemistry or by physical means, such as through ionic, covalent or other forces well known in the art ⁇ see, e.g., Dattagupta et al., Analytical Biochemistry 177:85-89 (1989); Saiki et al., Proc. Natl. Acad. ScL USA 86:6230-6234 (1989); Gravitt et al., J Clin. Micro. 36:3020-3027 (1998), each of which is hereby incorporated by reference in its entirety).
  • Either a terminal base or another base near the terminal base can be bound to the fluorescent label.
  • a terminal nucleotide base of the oligonucleotide probe can be modified to contain a reactive group, such as (without limitation) carboxyl, amino, hydroxyl, thiol, or the like.
  • the fluorescent label can be any fluorophore that can be conjugated to a nucleic acid and preferably has a photoluminescent property that can be detected and easily identified with appropriate detection equipment.
  • Exemplary fluorescent labels include, without limitation, fluorescent dyes, semiconductor quantum dots, lanthanide atom-containing complexes, and fluorescent proteins.
  • the fluorophore used in the present invention is characterized by a fluorescent emission maxima that is detectable either visually or using optical detectors of the type known in the art. Fluorophores having fluorescent emission maxima in the visible spectrum are preferred.
  • Exemplary dyes include, without limitation, Cy2TM, YO-PROTM- 1 ,
  • YOYOTM-1 Calcein, FITC, FluorXTM, AlexaTM, Rhodamine 110, 5-FAM, Oregon GreenTM 500, Oregon GreenTM 488, RiboGreenTM, Rhodamine GreenTM, Rhodamine 123, Magnesium GreenTM, Calcium GreenTM, TO-PROTM-1, TOTO ® -1, JOE, BODIPY ® 530/550, DiI, BODIPY ® TMR, BODIPY ® 558/568, BODIPY ® 564/570, Cy3TM, AlexaTM 546, TRITC, Magnesium OrangeTM, Phycoerythrin R&B, Rhodamine Phalloidin, Calcium OrangeTM, Pyronin Y, Rhodamine B, TAMRA, Rhodamine RedTM, Cy3.5TM, ROX, Calcium CrimsonTM, AlexaTM 594, Texas Red ® , Nile Red, YO-PROTM-3, YOYOTM-3, R-phyco
  • Attachment of dyes to the oligonucleotide probe can be carried out using any of a variety of known techniques allowing, for example, either a terminal base or another base near the terminal base to be bound to the dye.
  • 3'- tetramethylrhodamine may be attached using commercially available reagents, such as 3'-TAMRA-CPG, according to manufacturer's instructions (Glen Research, Sterling, Virginia).
  • Other exemplary procedures are described in, e.g., Dubertret et al, Nature Biotech. 19:365-370 (2001); Wang et al., J. Am. Chem. Soc, 125:3214-3215 (2003); Bioconjugate Techniques, Hermanson, ed. (Academic Press) (1996), each of which is hereby incorporated by reference in its entirety.
  • Exemplary proteins include, without limitation, both naturally occurring and modified (i.e., mutant) green fluorescent proteins (Prasher et al., Gene 111 :229-233 (1992); PCT Application WO 95/07463, each of which is hereby incorporated by reference in its entirety) from various sources such as Aequorea and Renilla; both naturally occurring and modified blue fluorescent proteins (Karatani et al., Photochem. Photobiol. 55(2):293-299 (1992); Lee et al., Methods Enzymol. (Biolumin. Chemilumin.) 57:226-234 (1978); Gast et al., Biochem. Biophys. Res. Commun.
  • Attachment of fluorescent proteins to the oligonucleotide probe can be carried out using substantially the same procedures used for tethering dyes to the nucleic acids, see, e.g., Bioconjugate Techniques, Hermanson, ed. (Academic Press) (1996), which is hereby incorporated by reference in its entirety.
  • Nanocrystal particles or semiconductor nanocrystals also known as
  • Quantum DotTM particles whose radii are smaller than the bulk exciton Bohr radius, constitute a class of materials intermediate between molecular and bulk forms of matter. Quantum confinement of both the electron and hole in all three dimensions leads to an increase in the effective band gap of the material with decreasing crystallite size. Consequently, both the optical absorption and emission of semiconductor nanocrystals shift to the blue (higher energies) as the size of the nanocrystals gets smaller.
  • capped nanocrystal particles of the invention are illuminated with a primary light source, a secondary emission of light occurs at a frequency that corresponds to the band gap of the semiconductor material used in the nanocrystal particles.
  • the band gap is a function of the size of the nanocrystal particle.
  • the illuminated nanocrystal particles emit light of a narrow spectral range resulting in high purity light.
  • Particles size can be between about 1 nm and about 1000 nm in diameter, preferably between about 2 nm and about 50 nm, more preferably about 5 nm to about 20 nm.
  • Fluorescent emissions of the resulting nanocrystal particles can be controlled based on the selection of materials and controlling the size distribution of the particles.
  • ZnSe and ZnS particles exhibit fluorescent emission in the blue or ultraviolet range (-400 nm or less);
  • Au, Ag, CdSe, CdS, and CdTe exhibit fluorescent emission in the visible spectrum (between about 440 and about 700 nm);
  • InAs and GaAs exhibit fluorescent emission in the near infrared range (-1000 nm), and PbS, PbSe, and PbTe exhibit fluorescent emission in the near infrared range (i.e., between about 700-2500 nm).
  • Preparation of the nanocrystal particles can be carried out according to known procedures, e.g., Murray et al., MRS Bulletin 26(12):985-991 (2001); Murray et al., IBMJ. Res. Dev. 45(l):47-56 (2001); Sun et al., J. Appl. Phys. 85(8, Pt. 2A): 4325-4330 (1999); Peng et al., J. Am. Chem. Soc.
  • nanocrystal particles can be purchased from commercial sources, such as Evident Technologies.
  • Attachment of a nanocrystal particle to the oligonucleotide probe can be carried out using substantially the same procedures used for tethering dyes thereto. Details on these procedures are described in, e.g., Bioconjugate Techniques, Hermanson, ed. (Academic Press) (1996), which is hereby incorporated by reference in its entirety.
  • Exemplary lanthanide atoms include, without limitation, Ce, Pr, Nd,
  • Attachment of a lanthanide atom (or a complex containing the lanthanide atom) to the oligonucleotide probe can be carried out using substantially the same procedures used for tethering dyes thereto. Details on these procedures are described in, e.g., Bioconjugate Techniques, Hermanson, ed. (Academic Press) (1996), which is hereby incorporated by reference in its entirety.
  • the fluorescent labels can be distinguished from one another using appropriate detection equipment. That is, the fluorescent emissions of one fluorescent label should not overlap or interfere with the fluorescent emissions of another fluorescent label being utilized. Likewise, the absorption spectra of any one fluorescent label should not overlap with the emission spectra of another fluorescent label (which may result in fluorescent resonance energy transfer that can mask emissions by the other label).
  • any of a variety of electrochemical or redox labels can be employed. Various electrochemical approaches to DNA detection have been developed (Palecek, E.
  • Exemplary electrochemical labels include, without limitation, a reporter group that contains a transition metal complex (e.g., ruthenium, cobalt, iron, or osmium complexes), or a redox moiety useful against an aqueous saturated calomel reference electrode (e.g., transition metal complexes, 1,4-benzoquinone, ferrocene, ferrocyanide, tetracyanoquinodimethane, N, N, N 1 , N'-tetramethyl-p- phenylenediamine, or tetrathiafulvalene), and redox moieties useful against an Ag/AgCI reference electrode (e.g., 9-aminoacridine, aciidine orange, aclarubicin, daunomycin, doxorubicin, pirarubicin, ethidium bromide, ethidium monoazide, chlortetracycline, tetracycline, minocycline
  • the electrochemical labels can optionally be linked through a suitable linker molecule, typically an organic moiety, as described in PCT Application WO 01/42508 to Choong et al., which is hereby incorporated by reference in its entirety.
  • a suitable linker molecule typically an organic moiety, as described in PCT Application WO 01/42508 to Choong et al., which is hereby incorporated by reference in its entirety.
  • the single-stranded oligonucleotide probe can be formed of either
  • RNA or DNA can contain one or more modified bases, one or more modified sugars, one or more modified backbones, or combinations thereof.
  • the modified bases, sugars, or backbones can be used either to enhance the affinity of the probe to a target nucleic acid molecule or to allow for conjugation to a fluorescent label.
  • Exemplary forms of modified bases are known in the art and include, without limitation, alkylated bases, alkynylated bases, thiouridine, and G-clamp (Flanagan et al., Proc. Natl. Acad. ScL USA 30:3513-3518 (1999), which is hereby incorporated by reference in its entirety).
  • modified sugars include, without limitation, LNA 5 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro (see, e.g., Freier and Attmann, Nucl. Acids Res. 25:4429-4443 (1997), which is hereby incorporated by reference in its entirety).
  • modified backbones include, without limitation, phosphoramidates, thiophosphoramidates, and alkylphosphonates. Other modified bases, sugars, and/or backbones can, of course, be utilized.
  • the single-stranded oligonucleotide probes can be of any length that is suitable to allow for rapid hybridization to target nucleic acids (if present) in the test solution, and rapid electrostatic association with negatively charged nanoparticles later introduced into the test solution. By rapid, it is intended that the single-stranded oligonucleotide probe can electrostatically associate with negatively charged nanoparticles at a rate that is greater (preferably by at least an order of magnitude) than the rate of association with other nucleic acids in the test solution prior to introduction of the oligonucleotide probe.
  • the single-stranded oligonucleotide probes are preferably between about 10 and about 50 nucleotides in length, more preferably between about 10 and 30 nucleotides in length, most preferably between about 12 and 20 nucleotides in length.
  • the single-stranded oligonucleotide probes can have their entire length or any portion thereof targeted to hybridize to the target nucleic acid. It is preferable for the oligonucleotide probe to have a nucleotide sequence that is 100 percent or perfectly complementary to part of the target nucleic acid sequence.
  • the amount of oligonucleotide probe introduced into the test solution can be determined based upon the total amount of negatively charged nanoparticles to be introduced into the hybridization solution and/or the total amount of target nucleic acid that is believed to be present.
  • the amount of oligonucleotide probe is at least slightly greater than the amount of negatively charged nanoparticles present in the hybridization solution (i.e., greater than a 1 : 1 ratio), more preferably greater than about 10:1, and up to about 30:1.
  • a reasonable match in the amounts of probe and target used are desirable for optimization of the assay. If the amount of nucleic acid in a sample can be reasonable estimated, then the ratio of probe:target should be between about 0.3:1 and about 3:1. If reasonable estimates cannot be made, then concentration series can be performed.
  • the relative concentrations of target and probe in the trial solution are not critical.
  • oligonucleotide probe can be synthesized using standard synthesis procedures or ordered from commercial vendors, such as Midland Certified Reagent Co.
  • the negatively charged nanoparticles can be formed of either a conductive metal or an uncharged substrate, such as glass.
  • the metal nanoparticles can be formed of any conductive metal or metal alloy that allows the nanoparticle to be capable of electrostatically associating with a single-stranded nucleic acid molecule or aggregating with other metal nanoparticles under appropriate conditions. (Prior to use in the present invention, it should be appreciated that the colloidal suspension maintains the metal nanoparticles in a stable environment in which they are substantially free of aggregation.) Importantly, the metal nanoparticles do not significantly associate electrostatically with hybridization complexes (that is, double-stranded nucleic acid molecules).
  • Exemplary metal nanoparticles include, without limitation, gold nanoparticles, silver nanoparticles, platinum nanoparticles, mixed metal nanoparticles (e.g., gold shell surrounding a silver core), and combinations thereof.
  • the metal nanoparticles can be magnetic, formed of a magnetic inner core such as cobalt and an outer core such as gold.
  • Suspensions of colloidal metal nanoparticles can be formed using the procedures described in Grabar et al., Anal. Chem. 67:735-743 (1995), which is hereby incorporated by reference in its entirety.
  • the metal nanoparticles in certain embodiments do not contain any ligands conjugated or otherwise bound to their outer surface.
  • the colloidal suspension preferably contains metal nanoparticles of between about 5 nm and about 500 nm, most preferably between about 10 nm and 30 nm.
  • the nanoparticle formed of an uncharged substrate is preferably charged using anions or polyanions.
  • the anions or polyanions can be coupled to the substrate (e.g., glass) using standard glass binding chemistry.
  • Exemplary anions include, without limitation, citrate, acetate, carbonate, dihydrogen phosphate, oxalate, sulfate, and nitrate.
  • Exemplary polyanions include, without limitation, poly(2- acrylamido-2-methyl-l-propanesulfonic acid), poly(acrylic acid), poly(anetholesulfonic acid), poly(anilinesulfonic acid), poly(sodium 4- styrenesulfonate), poly(4-styrenesulfonic acid), and poly(vinylsulfonic acid).
  • Other anions and polyanions can also be employed.
  • the detection of hybridization between probe and target can be achieved in one of several preferred approaches: a colorimetric approach, a fluorimetric approach, and a redox or radiation approach. Each has a distinct advantage over the other and can be employed as desired.
  • a colorimetric assay in which the probe can be unlabeled
  • the optical property of the hybridization solution is the visible color thereof.
  • the negatively charged nanoparticles are preferably the metal nanoparticles.
  • a color change of the hybridization solution can be brought about by inducing aggregation of the plurality of metal nanoparticles as illustrated in Figure 1.
  • the colorimetric assay is particularly useful when quantification is not necessary and where expensive detection equipment is unavailable. Detection of the color change in the hybridization solution can be carried out by naked eye observation of a user (i.e., the person performing the assay).
  • Aggregation will only occur if an insubstantial number of oligonucleotide probes has electrostatically associated with the metal nanoparticles. If a substantial number of oligonucleotide probes has electrostatically associated with the metal nanoparticles (on average greater than about one or two per nanoparticle), aggregation will be inhibited noticeably. Aggregation (color change) indicates that the target nucleic acid was present in the test solution. Induction of aggregation can be carried out by introducing a salt solution into the hybridization solution, with the salt being of sufficient concentration to alter the electrostatic properties of the metal nanoparticles, thereby promoting their aggregation.
  • the salt solution preferably comprises a Na + concentration of between about 0.01 and about 1 M, more preferably between about 0.1 and about 0.3 M.
  • the introduction of the salt solution to the hybridization medium can either be carried out simultaneously with the introduction of the solution containing the metal nanoparticles, or in succession therewith (either with or without a delay of up to about 15 minutes).
  • the colorimetric assay can be detected by naked eye observation, a user can either examine the hybridization solution for a detectable change in color or the assay can be carried out in parallel with one or more controls (positive or negative) that replicate the color of a comparable solution containing aggregated metal nanoparticles (negative control) and/or a comparable solution containing substantially non-aggregated metal nanoparticles (positive control).
  • the optical property of the hybridization solution is the fluorescence spectrum or the magnitude of a fluorescence peak by a f ⁇ uorophore. The photoluminescent property of the fluorophore label is detected after the hybridization procedure is allowed to proceed in the presence of the negatively charged nanoparticles.
  • Non-hybridizing oligonucleotide probes based on their size, will more rapidly associate electrostatically with the negatively charged nanoparticles than longer nucleic acid molecules in the hybridization solution.
  • the aggregates may or may not need to be separated from the nanoparticles remaining in solution.
  • separation is not required because the absence of hybridization (i.e., absence of the target) is indicated by substantial quenching of fluorescence by the fluorescent label when oligonucleotide probes electrostatically associate with one or more metal nanoparticles.
  • Hybridization between the oligonucleotide and the target nucleic acid molecule is indicated by a maintained photoluminescent property even after aggregation of the metal nanoparticles (which is achieved in the same manner as described above).
  • These alternatives are illustrated in Figure 2.
  • labeled probes remaining in solution i.e., in a ds- hybridization complex
  • aggregates to which ss-nucleic acids and probes have bound. Fluorescent emissions from the eluent (solution) - ⁇ y-
  • the fluorimetric assay is particularly useful for high sensitivity, when the target of interest is only one or many nucleic acid strands in a sample, when quantification of the target nucleic acid is desired, or when the presence of multiple distinct target nucleic acid molecules are being simultaneously analyzed within the same hybridization solution (i.e., using multiple oligonucleotide probes each with a distinct fluorophore attached thereto). Detection of the fluorescence properties of the hybridization solution can be achieved using appropriate detection equipment as is known in the art (e.g., fluorescence microscope, photomultipliers, CCD cameras, photodiodes, etc.).
  • the fluorimetric assay involves measuring fluorescence caused by the fluorophore(s) in the hybridization solution, a user can either examine the hybridization solution for the presence or absence of fluorescence. No controls are necessary.
  • the fluorimetric assay is highly sensitive to even small quantities and the photoluminescent properties can be detected with precise instrumentation, the fluorimetric assay lends itself to quantifying the amount of a target nucleic acid present in a test solution.
  • One approach for quantifying the amount of target nucleic acid present in the test solution involved comparing the results from the test solution to the results obtained from two control solutions that each contain known but differing amounts of the target nucleic acid. Thus, measurements of the photoluminescent property are obtained from the test solution and the two control solutions. Based on the photoluminescence of each solution, it is possible to calculate the quantity of the target nucleic acid in the test solution relative to the quantity of the target nucleic acid present in the first and second control solutions. Alternatively, the quantity of the target nucleic acid in the test solution can be calculated using the measured optical property (from the test solution) and a calibration curve of measured optical (e.g., photoluminescence) properties versus quantity of target nucleic acid.
  • the improvement described below resolves two limitations of the fiuorimetric assay described above.
  • the first limitation involves the contrast between unquenched fluorescence and fluorescence of hybridized probe. This can arise when the target to be hybridized represents a small enough fraction of the sample that it is overwhelmed by probe fluorescence that is not completely quenched. This can also arise if there were trace luminescence from the gold particles themselves.
  • the second limitation is that single (or very few) molecule sensitivity can be achieved when it is known that the fluorescent molecule is within a very limited area.
  • an improvement of the present invention relates to overcoming the limits of sensitivity of the fluorimetric assay described above.
  • the product of the hybridization procedure (which contains unbound ss-probe, ss-nontarget nucleic acid, and ds-target nucleic acid) is treated to allow for separation of the ds-target nucleic acid from the unbound ss-probe and ss-nontarget nucleic acid.
  • Exemplary approaches for treating the hybridization product for separating the ds-target nucleic acid include, without limitation: (1) the use of immobilized, electrostatically charged nanoparticles (e.g., citrate-coated gold or polyanion-coated glass); (2) causing electrostatically charged nanoparticles, with ss- nucleic acids bound thereto, to form insoluble aggregates (the so-called "crashout” approach); (3) concentrating ds-nucleic acid onto a charged solid surface (which can be performed alone or in combination with either of (1) or (2)); (4) the use of magnetic, electrostatically charged nanoparticles, which can be removed from solution with ss-nucleic acid adsorbed thereto; (5) the use of surfaces functionalized with thiol moieties to remove gold from solution; (6) addition of soluble dithiol or thioamine compounds to react with gold nanoparticles and remove them from solution via aggregation; or (7) mechanical methods to filter and remove the nanoparticles,
  • a modified approach for aggregation and separation involves the use of functionalized gold nanoparticles.
  • relatively large gold nanoparticles about 30 run up to about 100 nm, preferably about 40 to about 60 nm
  • whose surface is modified with mixed thiol self-assembled layers can be used.
  • Most of the surface can contain HS-(CH 2 ) n COOH to make the particle nominally water soluble and negatively charged so as not to adsorb ds-DNA.
  • a few sites per particle can be thiolated with HS-(CH 2 ) m SH dithiols that would allow for attachment to other gold nanoparticles, thereby forming aggregates that would crash out the gold/ss-DNA. It is preferably for m>n to facilitate the process. Though slow, the process should be effective in aggregating the gold nanoparticles.
  • Thiolated surfaces can also be prepared using of a variety of glass surfaces, e.g., a column of glass beads with an exposed thiol can be fabricated using standard silanization chemistry.
  • non-fluorescent labeling can be utilized, such as electrochemical or radioactive labeling using known (or hereafter developed) electrochemical or radioactive labels. These detection procedures can be used with separation, and preferably also with concentration of the ds-nucleic acid (carrying the probe). Electrochemical and radiation detection procedures are known in the art and can easily be adapted for detection of the labels, especially following separation protocols described above.
  • PCR can quickly amplify the total amount of nucleic acid in a sample, it is often used with hybridization-based detection procedures.
  • One of the significant benefits of the present invention is that the assay can be performed using the hybridization medium employed in the thermocycler. The only requirement, however, is that the product of PCR (typically a double-stranded cDNA) must be denatured prior to introducing the negatively charged nanoparticles. Specifically, the double-stranded cDNA can be denatured before or ⁇
  • Another important use of the assays of the present invention is for detecting a single nucleotide polymorphism ("SNP") in a target nucleic acid molecule. This is performed in slightly different manners depending on whether the colorimetric assay or the fluorimetric (or electrochemical or radiation) assay is to be performed. [0098] Basically, the colorimetric assay is performed in parallel using a test solution and a control solution.
  • the test hybridization solution contains a target nucleic acid molecule and at least one first single-stranded oligonucleotide probe having a nucleotide sequence that hybridizes to a region of the target nucleic acid molecule that may contain a SNP.
  • the probe contains a nucleotide sequence that does not hybridize perfectly to the region containing the SNP (i.e., no base-pairing occurs with the SNP).
  • the control hybridization solution contains the target nucleic acid molecule and at least one second single-stranded oligonucleotide probe including a nucleotide sequence that hybridizes perfectly to a region of the target nucleic acid molecule that does not contain a single-nucleotide polymorphism. Both the test and control hybridization solutions are then exposed to the metal nanoparticles, allowing any unhybridized probes in the hybridization solutions to electrostatically associate with the metal nanoparticles.
  • the hybridization solutions are maintained at a temperature that is between the melting temperature of the at least one first single-stranded oligonucleotide probe and the melting temperature of the at least one second single-stranded oligonucleotide probe (which has a higher melting temperature because it is perfectly complementary).
  • a determination is made whether an optical property of the test and control hybridization solutions are substantially different. A substantial difference indicates the presence of the single nucleotide polymorphism in the target nucleic acid molecule.
  • the first and second single-stranded oligonucleotide probes can possess the same nucleotide sequence (and be the same length) or a different nucleotide sequence. That is, the two oligonucleotide probes can hybridize to the same region of the target nucleic acids or different regions. If the latter, then the target nucleic acid molecule in the control solution is, e.g., a cDNA molecule that is known not to possess the particular SNP being detected in the test solution. If the former, then the hybridization region of the target nucleic acid molecule in the control solution is known to be stable and free of SNPs (i.e., contains a wild-type sequence).
  • the oligonucleotide probe for the control assay can be longer or can possess a modified structure (e.g., modified bases, backbone, etc.) that enhances the stability between the probe and target.
  • the fluorimetric assay is performed substantially as described above, except that the temperature of the hybridization solution is measured when quenching of photoluminescence from the fluorescent label begins (i.e., the temperature is slowly reduced until quenching begins). The measured temperature represents the melting temperature between the probe and the target nucleic acid.
  • This measured melting temperature is then compared to a known melting temperature of a perfectly complementary probe (this measurement can either be provided with a commercial kit or measured by performing the assay in parallel). A difference between the melting temperatures indicates the presence of the single nucleotide polymorphism in the target nucleic acid molecule.
  • these assays can also be performed when using the separation and detection procedures described above.
  • Yet another important use of the assays of the present invention is for detecting the presence of a pathogen in a sample. Basically, a sample is obtained
  • nucleic acid is isolated from the sample. Having isolated the nucleic acid, either RNA or DNA, an assessment can be made as to whether enough of the sample is present to afford detection using the assays or whether PCR or RT-PCR is necessary to amplify the isolated nucleic acid. Thus, amplification may or may not be necessary. For example, total RNA isolated from a sample may be of sufficient quantity to proceed without RT-PCR; whereas total DNA isolated from a sample may require amplification.
  • the assay of the present invention is performed and the optical property (color or fluorescence intensity) of the hybridization solution is measured or assessed to determine whether or not the single-stranded oligonucleotide probe has hybridized to the target nucleic acid, indicating presence of the pathogen.
  • This assay can also be performed when using the separation and detection procedures described above.
  • Yet another important use for the assays of the present invention is for genetic screening. Basically, a sample is obtained from a patient and nucleic acid is isolated from the sample. Because genetic screening will typically involve DNA isolation and analysis, it will typically (though not necessarily) require amplification.
  • the assay of the present invention is performed and the photoluminescent property of the hybridization solution is measured or assessed to determine whether or not the single-stranded oligonucleotide probe has hybridized to the target nucleic acid, indicating presence of a genetic marker for a genetic condition, a hereditary condition (e.g., paternity, maternity, relatedness, etc.), or identifying an organism.
  • This assay can also be performed when using the separation and detection procedures described above.
  • a further use of the assays of the present invention is detection of a protein or antibody in a sample.
  • Immuno-PCR is a procedure that can afford cDNA amplification only if a targeted protein is present in a sample.
  • the assays of the present invention can be coupled with the amplification detection procedure of immuno-PCR to confirm presence of the amplified cDNA in the hybridization medium and, thus, the target protein in a sample.
  • a sample is obtained and immuno-PCR is performed using the sample, wherein the immuno-PCR results in amplification of a nucleic acid that is conjugated to a protein.
  • the assays of the present invention are performed where the nucleic acid that is conjugated to the protein (or its complement) becomes the target of the colorimetric or fluorimetric assay of the present invention.
  • This assay can also be performed when using the separation and detection procedures described above.
  • a further use of the assays of the present invention is quantifying the amount of amplified nucleic acid prepared by polymerase chain reaction (or similar amplified procedure).
  • one or more, and preferably two or more fluorescently labeled oligonucleotide primers are provided that each have a nucleotide sequence capable of hybridizing to a nucleic acid molecule, or its complement, that us to be amplified.
  • Amplification using the primers is carried out using any of a variety of known amplification procedures (such as polymerase chain reaction) using a target nucleic acid molecule, and/or its complement, and the provided fluorescently labeled oligonucleotide primers.
  • the fluorimetric method of the present invention is performed on a sample obtained after the amplification procedure has been performed.
  • the level of fluorescence detected from the sample indicates the amount of primer that has been incorporated into an amplified nucleic acid molecule.
  • the amount of fluorescence from a given sample should increase due to the reduced rate at which longer nucleic acid electrostatically associate to the metal nanoparticles.
  • Unextended primers on the other hand, will rapidly associate with the metal nanoparticles, which results in quenching of fluorescence by labels attached thereto.
  • This assay can also be performed when using the separation and detection procedures described above.
  • kits that can be used to practice the assays of the present invention.
  • the kits can include, among other components, various containers that contain individual components that are used in accordance with the methods of the present invention, as well as instructions for carrying out one or more embodiments of the invention.
  • the kit includes a first container that contains a colloidal solution of metal nanoparticles, and a second container that contains an aqueous solution containing at least one single-stranded oligonucleotide probe having a nucleotide sequence that is substantially complementary to a target nucleic acid molecule.
  • the oligonucleotide probe in the second container may or may not be conjugated to a fluorescent label of the types described above.
  • the second container can optionally contain additional oligonucleotide probes (directed to the same or different target nucleic acid molecules), each having a distinct fluorescent emission pattern.
  • containers containing control solutions, salt solutions, and various instructions can also be provided.
  • the kit includes a first container that contains a colloidal solution of negatively charged nanoparticles, and a second container that contains an aqueous salt solution suitable to induce aggregation of the negatively charged nanoparticles.
  • This particular kit format is desired when the user intends to supply their own probe (with labels) and detection equipment. That is, depending upon the probes employed, detection devices suitable for electrochemical labels, radioactive labels, or fluorescence labels can be employed as desired.
  • the kit can optionally include a filter that is suitable to remove salt-induced aggregates while allowing passage of non-aggregated nanoparticles and ds-nucleic acids, as well as instructions for performing the assays of the present invention.
  • the kit includes a plurality of negatively charged nanoparticles bound to a substrate, for example, glass beads.
  • the substrate can be packed into a column, where they act as a filter to remove short, ss- nucleic acid while allowing ds-nucleic acid to flow through.
  • the kit can also include instructions for performing the assays of the present invention.
  • Example 1 Materials and Methods for Example 1 [0110] A colloidal solution of gold nanoparticles of about 13 nm diameter synthesized via citrate reduction OfHAuCl 4 (Grabar et al., Anal. Chem. 67:735-743 (1995), which is hereby incorporated by reference in its entirety) was used. The concentration of the colloidal solution was typically 17 nM. Lyophilized oligonucleotide sequences and their complements were purchased from MWG Biotech (High Point, NC) and dissolved in 10 mM phosphate buffer solution.
  • oligonucleotides purchased from MWG Biotech (High Point, NC) were used. Solutions in quartz cells with 1 cm path length were studied on a Jobin-Yvon Fluorolog-3 spectrometer with front face collection geometry and 4 nm resolution. Resonance Raman spectra were taken on these dye labeled oligonucleotides with steady state 532 nm excitation and detection by an Ocean Optics CCD array with a holographic notch filter to reject Rayleigh scattering. The resolution was approximately 10 cm "1 . Photographs were taken with a Canon S-30 digital camera.
  • Example 1 Gold Nanoparticles Preferentially Adsorb Single-Stranded Nucleic Acid Rather Than Double-Stranded Nucleic Acid
  • FIGS 4A-B DNA and gold nanoparticles is illustrated in Figures 4A-B.
  • dye tagged ss-DNA quenching of the dye photoluminescence and enhancement of resonant Raman scattering from the dye were observed. Both of these require intimate contact between the dye and the gold since they are effects of electronic interactions with the gold plasmons.
  • Figure 5 A presents spectra of the colloid prior to and after addition of ss-DNA or ds-DNA and salt/buffer solution.
  • Figure 5B illustrates a condensed form of the same data for two ss-
  • analyte solution contains a mixture of oligonucleotide sequences as might occur in products of polymerase chain amplification, where primers and other fragments are present (Rolfs et al., PCR: Clinical Diagnostics and Research, Springer- Verlag, Berlin Heidelberg (1992), which is hereby incorporated by reference in its entirety).
  • Figure 6 A illustrates the result for a mixed oligonucleotide analyte with various fractions of target sequence and it is clear that as little as 30% target is easily detected. A situation similar to concentration mismatch occurs when the target and probe sequences are complementary but have different lengths.
  • a 1 cm path length provides optical densities near unity. Empirically, it is easy to visually identify the colour in 5 ⁇ L droplets that contain less than 100 femtomoles of gold particles.
  • Figure 5B illustrates that ss-DNA concentrations only slightly greater than the nanoparticle concentration are sufficient to stabilize the colloid against aggregation when exposed to salt. Consequently, one would expect to be able to differentiate between amounts of ss- and ds-DNA of order 100 femtomoles without instrumentation.
  • the method is easily adapted to identifying single base pair mismatches between probe and target as is essential for detection of biologically important single nucleotide polymorphisms (Rolfs et al., PCR: Clinical Diagnostics and Research, Springer- Verlag, Berlin Heidelberg (1992), which is hereby incorporated by reference in its entirety). Utilized was the fact that the kinetics of ds- DNA dissociation into ss-DNA fragments depend on the binding strength (Owczarzy et al, Biopofymers 44:217-239 (1997); Santalucia et al., J. Am. Chem. Soc.
  • ds'-DNA mismatched ds-DNA
  • the ds-DNA from the trial solution was allowed to dehybridize briefly in water without salt before adding gold colloid and the salt/buffer solution.
  • the assay described has additional benefits beyond its speed and simplicity. Because of the ability to exploit the electrostatic properties of the DNA, hybridization is separated from detection so that the kinetics and thermodynamics of DNA binding are unperturbed by steric constraints associated with probe functionalized surfaces. In addition, the assay is homogeneous as it occurs exclusively in the liquid phase, a feature that makes it easy to automate using standard robotic manipulation of microwell plates. The ability to differentially adsorb ss-DNA onto the gold particles can also form the basis for a sensitive assay based on fluorescence that still avoids tagging of the analyte.
  • the fluorescence of the ss-DNA can be selectively quenched as in Figure 4A since it forces the dye to be near the gold nanoparticles where the fluorescence is quenched (Dubertret et al., Nature Biotechnol. 19:365-370 (2001); Du et al., J. Am. Chem. Soc. 125:4012-4013 (2003), which are hereby incorporated by reference in their entirety). If the tagged probe ss-DNA binds the target, however, the ds-DNA does not adsorb on the gold and the fluorescence persists.
  • Colloidal Au-np suspensions are stabilized against Au-np aggregation by adsorption of negatively charged ions that lead to strong electrostatic repulsion between the nanoparticles (Hunter, Foundations of Colloid Science. Oxford University Press Inc., NY (2001), which is hereby incorporated by reference in its entirety).
  • sodium citrate is added to gold nanoparticles during their synthesis so that citrate adsorption makes the Au-np surfaces negatively charged.
  • Both the colorimetric and fluorescent detection protocols take advantage of the rapid adsorption of single stranded oligonucleotides to the Au-np. This adsorption has been documented using fluorescence quenching and Raman experiments ⁇ see Examples infra).
  • oligonucleotides are themselves commonly regarded as negatively charged species presenting negatively charged phosphate backbones that would be repelled by citrate.
  • the rapid ss-DNA adsorption can be rationalized with a model where single stranded oligonucleotides can configure themselves with hydrophobic bases facing the Au-np. In this geometry, dipolar attraction can reduce the barrier to adsorption of ss-DNA and ss-RNA ⁇ see Examples infra).
  • Double stranded oligonucleotides are unable to achieve an uncoiled geometry with exposed bases and, therefore, experience much larger repulsion by the ions on the Au-np surface. Consequently, they take much longer times to adsorb or do not adsorb to the Au-np at all under some conditions.
  • the single stranded oligonucleotides add negative charge density to the Au-np surface and act to enhance the stability of the colloid. It is therefore possible to protect the colloid from aggregation upon exposure to amounts of salt that would ordinarily screen the electrostatic repulsion between Au-np and induce aggregation. Hence, the gold will remain pink upon exposure to salt following exposure to ss-DNA or ss-RNA, while it will turn grayish-blue following exposure to ds-DNA or ds-RNA. This observation forms the basis for the colorimetric hybridization assay. The preferential adsorption of short ss-DNA probe sequences on Au-np can also be exploited to perform the fluorescent assay. When the ss-DNA probe is fluorescently tagged, adsorption to the metallic surface results in fluorescence quenching (Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer ⁇
  • HAuCl 4 (Gradar et al., Anal. Chem. 67:735-743 (1995), which is hereby incorporated by reference in its entirety). Briefly, 500 mL of 1 mM HAuCl 4 was brought to a rolling boil with vigorous stirring. 50 mL of 38.8 mM sodium citrate was quickly added to the solution, and boiling was continued for 10 min. The heating mantle was then removed and the stirring was continued for an additional 15 minutes.
  • AU oligonucleotides were purchased from MWG Biotech, Inc. (High Point, NC) without further purification. Probes hybridize with targets in 10 mM phosphate buffer solution with 0.3 M NaCl for more than 5 minutes at room temperature or proper temperature.
  • Rhodamine red-labeled probe AGGAATTCCATAGCT (SEQ ID NO: 25); and Target nucleic acid: AGCTATGGAATTCCT (SEQ ID NO: 26).
  • Rhodamine red-labeled probe AGGAATTCCATAGCT (SEQ ID NO: 25);
  • Complementary Target A ACT AGGC ACTGTACGCCAGCTATGGAATTCCTT
  • Noncomplementary Target C TGTGTTGAACCTGGTGAAGTTGTAATCTGGAA
  • Rhodamine red-labeled probe AGGAATTCCATAGCT (SEQ ID NO: 25); Complementary Target A': ACTAGGC ACTGTACGCCAGCTATCGAATTCCT TAGCTATGAGATCCTTCG (SEQ ID NO: 27); and Complementary Target B' : GTTAGCTATGAGATCCTTCGTAGGCACTGTAC GCO4 GCTATCGAATTCCT (SEQ ID NO: 28).
  • Rhodamine red-labeled probe 1 CTGAATCCAGGAGCA (SEQ ID NO: 29); Complementary Target 1: the complement of probe 1; Cy5-labeled probe 2: TAGCTATGGAATTCCTCGTAGGCA (SEQ ID NO: 6); Complementary Target 2: the complement of probe 2; and Non-complement target: ATGGCAACTATACGCGCTAC (SEQ ID NO: 30).
  • a fraction of hybridized solution was added to 500 ⁇ L of 17 nM gold colloid solution, and an additional 500 ⁇ L of the 0.1 M saline 10 mM phosphate buffer solution was added if without specific illustration.
  • the fluorescence of this mixture was recorded immediately using either a fluorimeter, or a fluorescence microscope and camera. Fluorescence spectra were measured on a fluorimeter with excitation at 570 nm, and emission range from 585 to 680 nm, with slits set for 4 run bandpass unless specific illustration was given. Fluorescence images were recorded with a fluorescence confocol microscope equipped with notch filter and narrow bandpass interference filter. Fluorescence was excited by a 532 nm laser source.
  • Example 2 Differential Fluorescence Quenching of Dye-Tagged Single- Stranded DNA and Double-Stranded DNA
  • DNA oligonucleotides labeled with rhodamine red fluorescent dye covalently attached at the 5' end were used as probes.
  • Several microliters of ⁇ M solutions of probe were exposed to the target sequences for trial hybridization in 10 mM phosphate buffer with 0.3 M NaCl.
  • the hybridization solutions were added to colloidal gold suspensions and additional phosphate buffer saline solution was added to assist in stabilizing ds-DNA.
  • Figure 7A illustrates the result of a measurement comparing the photoluminescence from trial solutions with complementary and non-complementary targets. Fluorescence contrast larger than 100:1 was observed because unhybridized probes efficiently adsorb on the gold nanoparticles so that their fluorescence is quenched. The adsorption mechanism is entirely electrostatic, as discussed in Example 1 above. The adsorption and concomitant fluorescence quenching are irreversible. [0130] Addition of the trial hybridization solutions and salt to the gold colloid eventually cause aggregation of the colloid. The latter leads to precipitation so that the nanoparticles are no longer an effective quencher of the probe fluorescence.
  • Figure 7A illustrates that this is not necessary as long as the fluorescence measurements are made within about 15 minutes.
  • Figure 7B illustrates measuring the fluorescence of a very small aliquot of the solution containing only 0.1 femtomoles of target and this is easily detected with a fluorescence microscope and camera.
  • the assay can work to determine whether target strands are present even in complex mixtures of DNA oligonucleotides as demonstrated by the data of Figure 8B. In that case, 1% complementary target was mixed with 99% non-complementary target to verify the presence of the target sequence.
  • the tolerance of the assay to mixtures, along with its sensitivity, provides the potential for it to be used without target amplification by PCR.
  • Figure 9 shows the detection by one long target complementary to the probe in the middle portion and another long target complementary to the probe at one end.
  • This procedure should be applicable to rapid detection of single nucleotide polymorphisms in genomic DNA, an exciting prospect for eliminating time-consuming and expensive gel sequencing procedures that are currently the standard protocol (Rolfs et al., PCR: Clinical Diagnostics and Research, Springer- Verlag, Berlin Heidelberg (1992), which is hereby incorporated by reference in their entirety).
  • PCR Clinical Diagnostics and Research, Springer- Verlag, Berlin Heidelberg (1992)
  • the differential quenching assay can also be multiplexed to simultaneously look for several sequences on a single target or for several targets.
  • Figure 10 illustrates this where two different probes with two different dyes are hybridized with a mixture of targets. If spectroscopic detection is used, it is plausible to imagine expanding this approach to 5 or 6 targets with conventional dyes and even more with semiconductor nanoparticle fluorophores that have spectrally sharp emission. This, of course, presumes that these do not perturb the essential electrostatics that is the basis of the method.
  • the entire assay can be completed in less than 10 minutes because the hybridization step occurs in solution under optimized conditions and is separated from the detection step.
  • a sensitivity to less than 0.1 femtomole of DNA oligonucleotides has been demonstrated, but, because the method is nearly a null method and relies on fluorescence detection, it is probably possible to improve this by several orders of magnitude. It is believed that the method has enormous promise for applications to pathogen detection, clinical analysis of SNPs, and biomolecular research.
  • UV/VIS/NIR spectrometer Lambda 19 Quartz cells with 2 mm or 5 mm path length were used and water was used as reference. Fluorescence spectra and intensities versus time were recorded on a Jobin-Yvon Fluorolog-3 spectrometer with excitation at 570 nm and emission at 590 nm, each with slits set for 4 nm bandpass. Quartz cells with 1 cm path length and front face collection were used for the fluorescence measurements.
  • Example 7 Effects of Oligonucleotide Probe Length and Temperature on Adsorption of ss-DNA to Gold Nanoparticles
  • 300 ⁇ L gold colloid was mixed with 300 picomole 24 mer ss-DNA (5'-TGC CTA CGA GGA ATT CCA TAG CTA-3' (SEQ ID NO: 4)) in 10 ⁇ L of 10 mM PBS containing 0.2 M NaCl, and then 100 ⁇ L of 10 mM PBS containing 0.2 M NaCl was added.
  • ss-DNA sequences were 10 mer (5'- CAG GAA TTC C-3' (SEQ ID NO: 5)), 24 mer (5'-TAG CTA TGG AAT TCC TCG TAG GCA-3' (SEQ ID NO: 6)), and 50 mer (5'-GAA CCT CTG CTC AAC AAG TTC CAG ATT ACA ACT TCA CCA GGT TCA ACA CA-3' (SEQ ID NO: I)).
  • the color of gold colloid is very sensitive to the degree of aggregation of nanoparticles in suspension (Quinten et al., Surf. ScL 172:557 (1986); Lazarides et al., J. Phys. Chem. B 104:460 (2000); Storhoff et al., J. Am. Chem. Soc. 122:4640- 4650 (2000), which are hereby incorporated by reference in their entirety), and the aggregation can be easily induced with electrolytes such as salt (Hunter, Foundations of Colloid Science, Oxford University Press Inc., New York (2001); Shaw, Colloid and Surface Chemistry, Butterworth-Heinemann Ltd., Oxford (1991), which are hereby incorporated by reference in their entirety).
  • gold nanoparticles have surface plasma resonance absorption peak at 520 nm (Figure 1 IA: red) and appear pink (Figure 1 IA, inset: left vial). Immediate aggregation of the gold nanoparticles occurs when enough salt is added to screen the electrostatic repulsion between the ion-coated gold nanoparticles. The result is a broad featureless absorption spectrum (Figure 1 IA: blue) and blue-gray color ( Figure 1 IA, inset: middle vial) characteristic of the surface plasma resonance of gold nanoparticle aggregates (Quinten et al., Surf. Sd. 172:557 (1986); Lazarides et al., J.
  • Figure 1 ID illustrates the proof of principle for each of these assays with synthesized 50 base oligonucleotide targets and rhodamine-labeled 15 base probe.
  • KCNEl gene indicative of long QT syndrome was amplified in Promega PCR master mix (Promega, Madison, WI) with Tag DNA polymerase for 5 min at 95°C; 35 cycles of 30s at 95°C, 30s at 56 0 C and 30s at 72°C; 10 min at 72°C and then held at 4 0 C, yielding 189 bp PCR product.
  • the method takes less than ten minutes to verify amplification of the appropriate sequence and test for SNPs with the same thermal cycler used to do the PCR.
  • the protocol illustrated schematically in Figure 13A was followed.
  • Two ss-DNA probes were chosen with sequences complementary to the desired PCR product that have melting temperatures lower than the primers and add these to the PCR product solution.
  • the PCR amplified ds-DNA is dehybridized at 95°C to produce ss-DNA. These mixtures are annealed below the probe melting temperature so that the probes can hybridize with the PCR amplified sequence if it is present.
  • the unconsumed primers also bind to the PCR product since they have melting temperatures higher than those of the probes.
  • competition for binding locations from rehybridization of the PCR amplified complement is negligible since it is slower for steric reasons.
  • the salt in the hybridization solution causes immediate gold nanoparticle aggregation and a color change if the probes have hybridized to the amplified DNA target ( Figure 13B, left vial).
  • the probes adsorb to the gold nanoparticles and prevent aggregation ( Figure 13B, right vial).
  • Example 9 Sequence Detection and Single Base-Pair Mismatch Detection of PCR-Amplif ⁇ ed Target cDNA
  • 8 ⁇ L of unmodified PCR product was mixed with 6 ⁇ L of 1 ⁇ M probe solution containing either two complementary probes or two non-complementary probes in 10 mM PBS containing 0.3 M NaCl. After 5 minute denaturation at 95 0 C and 1 minute annealing at 5O 0 C, 60 ⁇ L gold colloid was added and photographs were taken.
  • probe sequences are as follows: 5'-CCT GTC TAA CAC CAC AG-3 ' (SEQ ID NO: 14) and 5'-CCA CAG CTT GGT CAG AA-3' (SEQ ID NO: 15) (complementary probes);
  • hybridization occurs under optimized conditions that can be regulated independent of the assay.
  • the assay has also been applied to clinical samples of genomic DNA that screen for SNPs associated with a hereditary cardiac arrhythmia known as long QT syndrome. It is believed that this approach can replace some traditional analytical methods for post-processing of PCR amplified DNA and that it will find broad application.
  • RNA target containing either one complementary probe or one non-complementary probe in 10 mM PBS and 0.3M NaCl solution.
  • RNA and gold colloid were added and photographs were taken.
  • the amount of RNA and gold colloid can be increased or decreased accordingly. In this case, a relatively large amount RNA and gold was used to measure visible spectra on regular spectrometer.
  • RNA probe AGGAAUUCCAUAGCU (SEQ ID NO: 21); perfect matched target: AGCUAUGGAAUUCCU (SEQ ID NO: 22); and non-complementary target: CGAUCACGAGAUCGA (SEQ ID NO: 23).
  • RNA probe AGGAAUUCCAUAGCU (SEQ ID NO: 21); perfect matched target: AGCUAUGGAAUUCCU (SEQ ID NO: 22); and single mismatched target: AGCUAUAGAAUUCCU (SEQ ID NO: 24).
  • an RNA probe can be used to effectively discriminate between a SNP and a wild-type sequence.
  • a detection protocol employing a capture antibody and a biotinylated detection antibody coupled via streptavidin to a biotinylated DNA molecule can be employed in detecting the presence of an antigen using standard immuno-PCR procedures. If the antigen is present, PCR will result in amplification of the biotinylated DNA molecule. Assuming the antigen was present, the amplified PCR product will be detected by colorimetric or fluorimetric detection methods described in the above examples.
  • citrate-coated gold nanoparticles prepared as described above were attached to the surface of glass beads, the beads were loaded into a column, and then the hybridization product was introduced into the column to collect the eluted solution (which contains the double stranded DNA labeled with fluorophores).
  • This approach effectively solved the contrast problem identified above. This process can be repeated more than once to optimize the results.
  • the detailed procedure included the following steps:
  • Cleaning glass beads Glass beads of 1 mm diameter were washed with piranha solution for 20 min, rinsed with clean water thoroughly, and then dried on a hot plate. 2. Coating glass beads with amino-group terminal molecules: glass beads were immersed in aminopropyl triethoxysilane (APTES) in toluene solution for 30 min, and then washed thoroughly with toluene. The APTES-modif ⁇ ed glass beads were then baked in an oven at 100 0 C. 3. Coating glass beads with gold nanoparticles: APTES-modified glass beads were immersed in gold colloid for 30 min, then rinsed with clean water thoroughly, dried on a hot plate, and cooled to room temperature for use.
  • APTES aminopropyl triethoxysilane
  • Small anion glass beads can be made by exposure of glass beads to aqueous solution containing the anions. Under appropriate conditions of temperature and pH, the glass surface will be effectively coated with the anions.
  • Polyanionic coatings of a wide variety of substrates can be accomplished by simply dipping substrates in polyelectrolyte solutions according to the methods of Shiratori and Rubner, "pH-dependent Thickness Behavior of Sequentially Adsorbed Layers of Weak Polyelectrolytes," Macromolecules 33(11):4213-4219 (2000), which is hereby incorporated by reference in its entirety.
  • Example 14 Formation of Patterned Charged Films on a Substrate
  • Patterned charged films to be used to concentrate ds-nucleic acid can be fabricated in accordance with the procedures described in Zhang et al., "Particle Assembly On Patterned “Plus/Minus” Polyelectrolyte Surfaces Via Polymer-On- Polymer Stamping," Langmuir 18(11):4505-4510 (2002), which is hereby incorporated by reference in its entirety.
  • Example 15 Separating Citrate-Coated Gold Nanoparticles (and ss-DNA
  • the crashout method involves first using the interactions in solution to adsorb the ss-DNA preferentially on the gold (or other negatively charged) nanoparticles, but then removing the nanoparticles and ss-DNA bound thereto, leaving the ds-DNA (target) to be analyzed. This is called the "crashout” method since it involves removing the nanoparticles from solution rather than removing the ss-DNA from solution.
  • the protocol for this method is similar to the fluorescence method described above.
  • the analyte was first hybridized against the fluorescently tagged probe with sequence complementary to the target (whose presence is being screened).
  • the hybridization solution was then introduced into gold colloid, and followed by the addition of salt solution.
  • the salt concentration should be provided within the range of about 0.1- 1 M, because too much salt will permit the repulsion of the nanoparticle coating to be screened so that ds-DNA will adsorb, whereas too little salt will not cause the gold to aggregate.
  • anion- or polyanion-coated non-metallic nanoparticles can be substituted for the colloidal gold nanoparticles in the crash out method given that quenching of fluorescence is no longer relied upon for signal detection perse.
  • immobilized beads and crashout methods solve the contrast problem, but they also allow for the use of other labels besides fluorescent tags.
  • Two suitable labels are radioactive tags and electrochemical (“redox”) tags.
  • electrochemical detection can be carried out using either cyclic voltammetry (De-los- Santos-Alvarez, Anal. Chem. 74:3342-3347 (2002), which is hereby incorporated by reference in its entirety), stripping potentiometry (Wang et al., Anal. Chem.
  • the part of the analyte that comes through the column can be easily analyzed for fluorescence (or radioactivity or electrochemical activity).
  • fluorescence or radioactivity or electrochemical activity
  • the charged spot can be prepared using a polycation (e.g., polyamine), and detection equipment (e.g., fluorescence microscope) can be focused on the predefined location.
  • detection equipment e.g., fluorescence microscope
  • the charged spot can be a micro-electrode (e.g., gold, platinum, etc.) functionalized with a monolayer whose end group is positively charged, e.g., NH 2 .
  • a patterned polyelectrolyte can be made as follows. First, a negatively charged surface to which DNA will not adhere is formed on a glass slide by standard electrostatic self-assembly techniques. For example, a multilayer structure can be formed with a PAA (polyacrylic acid) top layer. A PDMS stamp can be fabricated with a predefined recessed region the size of the desired microscope focus. The stamp is inked with a monolayer to pattern that surface to be hydrophobic everywhere except for the place where the stamp is recessed. An ink made of ODA (CH 3 (CHa) 17 NH 2 ) in an organic solvent is suitable for this purpose; the applicants have previously demonstrated use of this ink.
  • ODA CH 3 (CHa) 17 NH 2
  • the amine is attracted to the carboxylate terminations of the PAA surface, thereby transforming the hydrophilic PAA to hydrophobic in the region where the ink layer is applied.
  • a positively charged electrolyte can be applied to the resulting patterned surface, and the electrolyte will only stick where there is no ODA.
  • the ODA can then be removed by rinsing with organic solvent, leaving the patterned charged surface.
  • Application of the processed analyte, where only tagged ds-nucleic acid should remain, will allow the DNA to be concentrated onto the positively charged spot for analysis.
  • Example 18 Formation of Positively Charged Microparticles
  • Polycationic polystyrene or silica microspheres can be made by exposure of microspheres to aqueous solutions containing cations. Under proper conditions of temperature and pH, the microsphere surface will effectively be coated with the cations. Polycationic coatings of a wide variety of substrates can be accomplished by simply dipping substrates in polyelectrolyte solutions.
  • Polycationic microparticles can be introduced into eluent that comes through a separation column of the type described in Example 17 above.
  • the microparticles will adsorb labeled ds-DNA onto a small volume.
  • the microparticles can be introduced onto a negatively biased electrode for analysis using a confocal microscope.
  • Gold colloid an aqueous suspension of Au-nps stabilized against aggregation by sodium citrate, was prepared as described elsewhere (Grabar et al., "Preparation and Characterization of Au Colloid Monolayers," Anal. Chem. 67:735-743 (1995), which is hereby incorporated by reference in its entirety). Briefly, 250 mL of 1 mM HAuCl 4 (Alfa Aesar, Ward Hill, MA) aqueous solution was heated to its boiling point while stirring.
  • RNA oligonucleotide targets and probes A 2- o-methyl RNA oligonucleotide (5'-AGG AAU UCC AUA GCU-3 ', SEQ ID NO: 34) was synthesized and purified by IDT (Coralville, IA) to be used as a probe sequence for the colorimetric assay. Three RNA sequences with the same length as the probe were used as targets. These were synthesized and purified (RNase-free HPLC purification, RNA oligos of greater than 85% foil length product) by IDT.
  • c-target One sequence (c-target) was complementary to probe, the second (mc-target: 5'-AGC UAU AGA AUU CCU-3 ', SEQ ID NO: 35) had a one base-pair mismatch with the probe and the third (nc-target: 5'-CGA UCA CGA GAU CGA-3', SEQ ID NO: 33) is not complementary to the probe.
  • rhodamine red labeled DNA were used as probes (wild-type probe: rhodamine red-5'-AGG AAT TCC ATA GCT-3', SEQ ID NO: 8, and mutant probe: rhodamine red-5'-AGG AAT GCC ATA GCT-3', SEQ ID NO: 36).
  • Rhodamine red labeled DNA sequences were purchased from MWG Biotech (High Point, NC). 2'-ACE protected 50 mer RNA50a and RNA50b were purchased from DHARMACOM RNA Technologies (Lafayette, CO). These two sequences only have a single base difference in their sequences (RNA50a (/RNA50b): 5'-ACU AGG CAC UGU ACG CCA GCU AUG GA(/C)A UUC CUU AGC UAU GAG AUC CUU CG-3', SEQ ID NOS: 31-32, respectively. RNA50a contains a sequence perfectly matched with the wild-type probe while the analogous segment of RNA50b has a single base-pair mismatch with the wild-type probe.
  • RNA and DNA solutions with concentrations of salt and phosphate buffer as specified in the text were made.
  • the requisite potassium phosphate (monobasic, anhydrous 99.999%) and sodium phosphate (dibasic, anhydrous, 99.999%) were obtained from Aldrich Chemical (Milwaukee, WI) and used as supplied.
  • Sodium chloride crystals were purchased from Mallinckrodt (Hazelwood, MO).
  • the trial solution was heated to 95°C for 3 minutes and then cooled to an appropriate temperature for the desired assay for 1 minute.
  • the temperature used for simple sequence detection was typically ambient while single base mismatch detection requires that hybridization takes place at a temperature between the melting temperature of the mismatch and that of the perfect match.
  • the gold colloid was used at ambient temperature regardless of the temperature of the trial solution.
  • Fluorescence Detection 5 ⁇ L trial hybridization solution was added to 500 ⁇ L gold colloid, then mixed with 500 ⁇ L of 10 PBS containing 0.3 M NaCl. The fluorescence spectrum of the mixture was recorded within 2 minutes after mixing in a fluorimeter (Fluorolog 3, Jobin Yvon) with excitation at 570 ran over the range of emission wavelengths from 585 to 680 nm. Spectrometer slits were set for 4 nm bandpass. Traces of photoluminescence versus time were recorded with at 590 nm near the rhodamine emission maximum.
  • FIGS 19A-D are images taken immediately after mixing trial hybridization solutions with gold colloid. The quantity of salt in the hybridization solution was adequate to cause Au-np aggregation in the absence of RNA. Each vial contains 10 ⁇ L trial hybridization solution that contains 10 mM PBS and 0.3 M NaCl and 50 ⁇ L gold colloid. In the hybridization solution, there were 20 picomoles of RNA probe and target.
  • RNA 2'-o-methyl RNA was used because of its high stability (Majlessi et al., "Advantages of 2'-O-methyl Oligoribonucleotide Probes for Detecting RNA Targets," Nucleic Acids Res. 26:2224-2229 (1998), which is hereby incorporated by reference in its entirety).
  • Complementary (c), single base mismatched (mc), and unrelated (nc) target sequences were used in the left, center and right vials, respectively. All trial hybridization solutions were heated at 95 0 C for 3 minutes, then annealed for 1 minutes at the specified temperatures (A: 20°C, B: 50°C, C: 59°C and D: 64°C).
  • Example 21 Fluorescent Detection of RNA Long-mers
  • colorimetric detection There are some limitations on colorimetric detection that can be ameliorated by using the fluorescent assay described above. Since traditional absorption spectroscopy is, by its nature, not a null experiment, its sensitivity is limited. Moreover, a number of ambiguities arise in the context of using the colorimetric method illustrated in the preceding examples. For example, it is easy to imagine circumstances where the quantities of target and probe differ so that the trial hybridization solution contains both single and double strands, hi addition, situations where the length of the probe does not match that of the target leaves a single stranded overhang on a double stranded complex. Using the fluorescent assay, these practical difficulties do not arise.
  • RNA sequence detection DNA sequences were labeled with rhodamine red as probes because RNA oligonucleotides are difficult to fluorescently label.
  • Long synthetic targets 50 bases
  • 15 base probes were used to assay for a complementary sequence on the targets.
  • the hybridization solution was heated to 94 0 C for 3 minutes to break up secondary structure and annealed for 1 minute at a lower temperature.
  • single base mutations can be detected by careful choice of annealing temperature for hybridization.
  • the duplex formed from a probe and mutant target has a lower melting temperature than the duplex formed from the probe and wild-type target.
  • Figure 22 depicts the time course of the luminescence after mixing the trial hybridization solution with Au-nps as monitored at the wavelength maximum of the fluorescence in an experiment analogous to that of Figure 21, but where the hybridization solution contains 5 picomoles probe, 5 picomoles target and 50 picomoles of short RNA noncomplementary segments.
  • the hybridization solution contains 5 picomoles probe, 5 picomoles target and 50 picomoles of short RNA noncomplementary segments.
  • Each combination of wild type and mutant probe and targets are illustrated at an annealing temperature below the wild type melting temperature.
  • Figure 22 An important implication of Figure 22 is that the fluorescent assay can tolerate substantial amounts of RNA degradation into short sequences as often occurs. As long as there is an adequate concentration of Au-np, these do not interfere with adsorption of unhybridized probes and the attendant fluorescence quenching essential to the assay. [0189]
  • the dynamics reflected in Figure 22 are important in the performance of the assay since the fluorescence should be evaluated at a time long enough to allow for adsorption of the unhybridized probes but short enough relative to the lifetime or adsorption rate of the hybridized complex formed between probe and target. Under the conditions of Figure 22, the adsorption of unhybridized probes on the Au-np is very rapid and occurs prior to the beginning of the trace.
  • the subsequent slow decay seen in Figure 22 has several possible explanations.
  • the complex formed between the probe and target may not be perfectly stable in the gold colloid and may slowly dehybridize. Even if it does not, single stranded portions of the long target strand may adsorb and bring the probe fluorophore close to the Au-np so that quenching is observed.
  • the above examples demonstrate a simple approach to detection of specific RNA sequences based on the differential adsorption rates for single and double stranded oligonucleotides onto Au-nps.
  • a colorimetric assay for target RNA sequences with 2-o-methyl RNA probes and a fluorescent assay based on hybridization of target RNA with fluorescently labeled DNA probes have been developed.
  • the assays require only commercially available reagents.
  • a key strength of the methods is that the hybridization step is completed independent of the assay so that it can be performed under optimal conditions for rapid, efficient hybridization. Each assay therefore takes less than 10 minutes so that issues concerning RNA instability are minimized. Single base mismatches between probe and target sequences are easily detected with high contrast.
  • the fluorescent assay is particularly promising since it is effective even for complex target mixtures and in cases where the probe and target have quite different length. This will allow its use in searching for target sequences in samples of genomic RNA.

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Abstract

Methods and kits are provided for detecting the presence or absence of target nucleic acid sequences in a sample. The methods and kits involve the use of negatively charged nanoparticles and the electrostatic interactions between the metal nanoparticles and nucleic acid molecules. The methods rely upon the differential interaction of ss-nucleic acids and ds-nucleic acids with the negatively charged nanoparticles that differentiate between tagged oligonucleotide probes that hybridize with a target and those that do not. Improvements in sensitivity for a fluorescent variation of the method have been obtained by including a step of separating the ds-nucleic acids in solution from the negatively charged nanoparticles to which ss-nucleic acids have been bound, and then detecting for the presence of the ds-target nucleic acids in the solution. The same separation protocols can be used to make the detection approach viable with electrochemical or radioactive tags.

Description

METHODS FOR SEPARATING SHORT SINGLE-STRANDED NUCLEIC
ACID FROM LONG SINGLE- AND DOUBLE-STRANDED NUCLEIC ACID,
AND ASSOCIATED BIOMOLECULAR ASSAYS
[0001] This application is a continuation-in-part of U.S. Patent Application
Serial No. 10/847,233, filed May 17, 2004, which claims the priority benefit of U.S. Provisional Patent Applications Serial Nos. 60/471,257, filed May 16, 2003, and 60/552,793, filed March 12, 2004. This application also claims the priority benefit of U.S. Provisional Patent Application Serial No. 60/645,821, filed January 21, 2005. Each of the above-identified priority applications is hereby incorporated by reference in its entirety.
[0002] The present invention was made at least in part with funding received from the National Institutes of Health under grant AGl 8231. The U.S. government may retain certain rights in this invention.
FIELD OF THE INVENTION
[0003] The present invention relates to hybridization-based nucleic acid detection procedures and materials for practicing the same.
BACKGROUND OF THE INVENTION
[0004] Detection of specific oligonucleotide sequences is important for clinical diagnosis, biochemical and medical research, food and drug industry, and environmental monitoring, pathology and genetics (Primrose et al., Principles of Genome Analysis and Genomics, Blackwell Publishing, Maiden, MA, Third edition (2003); Hood et al., Nature 421 :444-448 (2003); Rees, Science 296:698-700 (2002)). Present assays are dominated by chip based methodologies (Epstein et al., Analytica Chimica Acta 469:3-36 (2002); Chee et al., Science 274:610-614 (1996)) that have two principal disadvantages. First, target labeling is usually required. Second, hybridization to sterically constrained probes on surfaces is slow. Approaches such as sandwich assays (Elghanian et al., Science 277:1078-1081 (1997); Taton et al.,
Science 289:1757-1760 (2000); Cao et al., Science 297:1536-1540 (2002); Park et al., Science 295:1503-1506 (2002)), immobilized molecular beacons (Dubertret et al., Nat. Biotech. 19:365-370 (2001); Du et al., J. of Am. Chem. Soc. 125:4012-4013 (2003)), surface plasmon resonance (Brockman et al., Annual Review of Physical Chemistry 51:41-63 (2000)), porous silicon microcavity emission (Chan et al., Materials Science & Engineering C-Biomimetic and Supramolecular Systems 15:277- 282 (2001)), and reflective interferometry (Lin et al., Science 278:840-843 (1997); Pan et al., Nano Lett. 3:811-814 (2003)) avoid the former problem, but still require complex surface attachment chemistry for probe immobilization and may suffer from slow response, hi several of these cases, a nontrivial rinse step is required to remove unbound target or a second hybridization step is required in the assay. [0005] Nearly all assays for DNA sequences use the polymerase chain reaction ("PCR") to amplify specific sequence segments from as little as a single copy of DNA to easily detected quantities (Reed et al., Practical Skills in Biomolecular Sciences, Addison Wesley Longman Limited, Edinburgh Gate, Harlow, England (1998); Walker et al., Molecular Biology and Biotechnology, The Royal Society of Chemistry, Thomas Graham House, Cambridge, UK (2000)). The use of PCR not only addresses sensitivity issues, but also effectively purifies samples to ameliorate the effects of large quantities of DNA that may not be of interest for a given assay. These features presently make the use of PCR nearly indispensable for the analysis of genomic DNA in spite of the development of a wide variety of innovative sensing approaches such as surface plasmon resonance ("SPR") (Thiel et al., Anal. Chem. 69:4948-4956 (1997); Jordan et al., Anal. Chem. 69:4939-4947 (1997); Nelson et al., Anal. Chem. 73:1-7 (2001); He et al., J. Am. Chem. Soc. 122:9071-9077 (2000)), fluorescent microarrays (Sueda et al., Bioconjugate Chem. 13:200-205 (2002); Paris et al., Nucleic Acids Res. 26:3789-3793 (1998); Lepecq et al., MoI. Biol. 27:87-106 (1967)), assays based on semiconductor or metal nanoparticles (Bruchez et al., Science 281 :2013-2016 (1998); Gerion et al., J. Am. Chem. Soc. 124:7070-7074 (2002); Chan et al., Science 281:2016-2018 (1998); Elghanian et al., Science 277:1078-1081 (1997); Taton et al., Science 289:1757-1760 (2000); Park et al., Science 295:1503-1506 (2002); Cao et al., Science 297:1536-1540 (2002); Maxwell et al., J. Am. Chem. Soc. 124:9606-9612 (2002); Dubertret et al., Nat. Biotech. 19:365- 370 (2001); Sato et al., J. Am. Chem. Soc. 125:8102-8103 (2003)), and water-soluble conjugated polymer based sensors (Gaylord et al., J. Am. Chem. Soc. 125:896-900 (2003)). These techniques have been demonstrated mostly on purified synthesized oligonucleotides, but it maybe possible to adapt some of them to be compatible with PCR amplified samples. Once PCR amplification is utilized, however, the merit of an assay is primarily determined by its simplicity rather than its sensitivity since additional amplification is straightforward. Most of the above approaches, as noted, require expensive instrumentation or involve time-consuming synthesis to modify DNA, substrates, or nanoparticles. In addition, it is usually necessary to conduct hybridization in the presence of substrates that introduce steric hindrance, leading to slow and inefficient binding between probe and target. As a result, post-processing of PCR amplified samples can be expensive and time-consuming (Rolfs et al., PCR: Clinical Diagnostics and Research, Springer- Verlag, Berlin Heidelberg (1992)). [0006] Complexes between DNA and negatively charged gold nanoparticles have been studied for many years (Mirkin et al., Nature 382:607-609 (1996); Alivisatos et al., Nature 382:609-611 (1996)), and many creative schemes have exploited gold nanoparticles covalently functionalized with DNA sequences to bind specific target DNA sequences, either for nano-assembly or for oligonucleotide sensing (Elghanian et al., Science 277: 1078-1081 (1997); Taton et al., Science
289:1757-1760 (2000); Park et al., Science 295:1503-1506 (2002); Cao et al., Science 297:1536-1540 (2002); Maxwell et al., J. Am. Chem. Soc. 124:9606-9612 (2002); Dubertret et al., Nat. Biotech. 19:365-370 (2001); Sato et al., J. Am. Chem. Soc. 125:8102-8103 (2003); Mirkin et al., Nature 382:607-609 (1996); Alivisatos et al., Nature 382:609-611 (1996); Chakrabarti et al., J. Am. Chem. Soc. 125:12531-12540 (2003); Loweth et al, Angew. Chem. Lit. Ed. 38:1808-1812 (1999); Mbindyo et al., Adv. Mαter. 13:249-254 (2001)).
[0007] Based on the foregoing, it would be desirable to provide an assay that utilizes charged nanoparticles and target nucleic acid molecules that require no modification for detection of the target nucleic acid. Moreover, it would be desirable to provide an assay where hybridization is completely separate from detection so that it can be performed under optimal conditions without steric constraints of surface bound probes that slow hybridization dramatically and make it less efficient. [0008] The present invention is directed to achieving these objectives and overcoming these and other deficiencies in the art. -A-
SUMMARY OF THE INVENTION
[0009] A first aspect of the present invention relates to a method for detecting presence or absence of a target nucleic acid molecule in a test solution (e.g., sample). This method includes the steps of: combining at least one single-stranded oligonucleotide probe with a test solution potentially including a target nucleic acid to form a hybridization solution, wherein the at least one single-stranded oligonucleotide probe and the test solution are combined under conditions effective to allow formation of a hybridization complex between the at least one single-stranded oligonucleotide probe and any target nucleic acid present in the test solution; exposing the hybridization solution to a plurality of metal nanoparticles under conditions effective to allow the at least one single-stranded oligonucleotide probe that remains unhybridized after said combining to associate electrostatically with the plurality of metal nanoparticles; and determining whether the at least one single-stranded oligonucleotide probe has hybridized to target nucleic acid or electrostatically associated with one or more of the plurality of metal nanoparticles, wherein hybridization to the target nucleic acid or electrostatic association with one or more metal nanoparticles is indicated by an optical property of the hybridization solution. [0010] There are several embodiments for this aspect of the invention that are particularly preferred. One embodiment, designated a colorimetric assay, utilizes an unlabeled oligonucleotide probe and involves making the determination by detecting a color change of the hybridization solution after the step of exposing, whereby a color change indicates substantial aggregation of the plurality of metal nanoparticles in the presence of the target nucleic acid. If no color change (or an insignificant change) occurs, absence of the target nucleic acid is indicated. Another embodiment utilizes a fluorescently labeled oligonucleotide probe and involves determining whether or not fluorescence can be detected following exposure to the plurality of metal nanoparticles, whereby elimination of fluorescence indicates absence of a target nucleic acid and remaining fluorescence indicates its presence. If fluorescence by the labeled oligonucleotide probes remains, the oligonucleotide probes have formed duplexes and remain dissociated from the metal nanoparticles (i.e., no fluorescence quenching has occurred). [0011] A second aspect of the present invention relates to a method for detecting a single nucleotide polymorphism ("SNP") in a target nucleic acid molecule. This method is carried out by combining (i) a test solution including a target nucleic acid molecule and (ii) at least one first single-stranded oligonucleotide probe that has a nucleotide sequence that hybridizes to a region of the target nucleic acid molecule that may contain a single-nucleotide polymorphism, to form a test hybridization solution, wherein said combining is carried out under conditions effective to allow hybridization between the target nucleic acid molecule and the at least one first single-stranded oligonucleotide probe to form at least one hybridization complex; combining (i) a control solution including the target nucleic acid molecule and (ii) at least one second single-stranded oligonucleotide probe that has a nucleotide sequence that hybridizes perfectly to a region of the target nucleic acid molecule that does not contain a single-nucleotide polymorphism, to form a control hybridization solution, wherein said combining is carried out under conditions effective to allow hybridization between the target nucleic acid molecule and the at least one second single-stranded oligonucleotide probe to form at least one hybridization complex; exposing the test and control hybridization solutions, while maintaining the hybridization solutions at a temperature that is between the melting temperature of the at least one first single-stranded oligonucleotide probe and the melting temperature of the at least one second single-stranded oligonucleotide probe, to a plurality of metal nanoparticles under conditions effective to allow unhybridized probes in the hybridization solutions to electrostatically associate with the metal nanoparticles; and determining whether an optical property of the test and control hybridization solutions are substantially different, indicating the presence of the single nucleotide polymorphism in the target nucleic acid molecule.
[0012] A third aspect of the present invention relates to a method for detecting a SNP in a target nucleic acid molecule. This method is carried out by: combining (i) a solution including a target nucleic acid molecule and (ii) at least one first single- stranded oligonucleotide probe having a nucleotide sequence and a fluorescent label attached thereto, wherein the nucleotide sequence hybridizes to a region of the target nucleic acid molecule that may contain a single-nucleotide polymorphism, to form a hybridization solution, wherein said combining is carried out under conditions effective to allow hybridization between the target nucleic acid molecule and the at least one first single-stranded oligonucleotide probe to form at least one hybridization complex; exposing the hybridization solution to a plurality of metal nanoparticles under conditions effective to allow unhybridized probes in the hybridization solution to electrostatically associate with the metal nanoparticles; determining a temperature of the hybridization solution where quenching of the photoluminescence by the fluorescent label begins, said temperature representing the melting temperature; and comparing the melting temperature for the hybridization solution with a known melting temperature of a perfectly complementary probe, wherein a difference between the melting temperatures indicates the presence of the single nucleotide polymorphism in the target nucleic acid molecule.
[0013] A fourth aspect of the present invention relates to a method for detecting a target nucleic acid in a test solution. This method includes the steps of: subjecting a portion of a test solution potentially including a target nucleic acid to polymerase chain reaction and obtaining a product solution that includes single- stranded nucleic acid products of the polymerase chain reaction; combining at least one single-stranded oligonucleotide probe with the product solution to form a hybridization solution under conditions effective to allow formation of a hybridization complex between the at least one single-stranded oligonucleotide probe and any target nucleic acid present in the product solution; exposing the hybridization solution to a plurality of metal nanoparticles under conditions effective to allow any single- stranded nucleic acids in the hybridization solution to associate with the plurality of metal nanoparticles; and determining whether the at least one single-stranded oligonucleotide probe has hybridized to target nucleic acid or electrostatically associated with one or more of the plurality of metal nanoparticles, wherein hybridization to the target nucleic acid or electrostatic association with one or more metal nanoparticles is indicated by an optical property of the hybridization solution. [0014] A fifth aspect of the present invention relates to a method of detecting a pathogen in a sample that includes the steps of obtaining a sample that may contain nucleic acid of a pathogen, and then performing a method of the present invention using an oligonucleotide probe specific for a target nucleic acid of the pathogen, wherein the step of determining that the at least one single-stranded oligonucleotide probe has hybridized to the target nucleic acid indicates presence of the pathogen. [0015] A sixth aspect of the present invention relates to a method of genetic screening. This method is carried out by obtaining a sample, isolating DNA from the sample, amplifying the DNA isolated from the sample, and then performing a method of the present invention using an oligonucleotide probe specific for diagnosing a genetic condition, hereditary condition, or the like, wherein the step of determining that the at least one single-stranded oligonucleotide probe has hybridized to the target nucleic acid indicates predisposition to the genetic condition, hereditary condition, or identification of an organism. [0016] A seventh aspect of the present invention relates to a method of detecting a protein in a sample. This method is carried out by obtaining a sample, performing an immuno-polymerase chain reaction procedure using the sample, wherein the immuno-polymerase chain reaction procedure results in amplification of a nucleic acid that is conjugated to a protein, and then performing a method of the present invention using an oligonucleotide probe specific for the nucleic acid that is conjugated to the protein (or its complement), wherein the step of determining that the at least one single-stranded oligonucleotide probe has hybridized to the target nucleic acid indicates that the protein is present in the sample.
[0017] An eighth aspect of the present invention relates to a method of quantifying the amount of amplified nucleic acid prepared by polymerase chain reaction. This method is carried out by providing two or more fluorescently labeled oligonucleotide primers that each have a nucleotide sequence capable of hybridizing to a nucleic acid molecule, or its complement, to be amplified; performing polymerase chain reaction using a target nucleic acid molecule and/or its complement, and the provided fluorescently labeled oligonucleotide primers; and performing the fluorimetric method of the present invention on a sample obtained after said performing polymerase chain reaction, wherein the level of fluorescence detected from the sample indicates the amount of primer that has been incorporated into an amplified nucleic acid molecule. [0018] A ninth aspect of the present invention relates to a method for detecting presence or absence of a target nucleic acid in a test solution that includes the steps of: combining at least one single-stranded oligonucleotide probe with a test solution potentially including a target nucleic acid to form a hybridization solution, wherein the at least one single-stranded oligonucleotide probe and the test solution are combined under conditions effective to allow formation of a hybridization complex between the at least one single-stranded oligonucleotide probe and any target nucleic acid present in the test solution; exposing the hybridization solution to a plurality of negatively charged nanoparticles under conditions effective to allow any single- stranded oligonucleotide probe or non-target nucleic acid that remains unhybridized after said combining to associate electrostatically with the plurality of negatively charged nanoparticles; separating the plurality of negatively charged nanoparticles from the hybridization solution after said exposing; and determining whether the at least one single-stranded oligonucleotide probe has hybridized to target nucleic acid. This method can be adapted for SNP detection, detection of PCR products, detection of pathogen nucleic acids, and quantification of target nucleic acids in accordance with the other aspects of the present invention.
[0019] A tenth aspect of the present invention relates to kits containing various components that will allow a user to perform one or more methods of the present invention. According to one embodiment, the kits minimally include a first container that contains a plurality of negatively charged nanoparticles; and a second container that contains a salt solution having a concentration of salt that is effective to cause aggregation of the negatively charged nanoparticles. According to a second embodiment, the kits can further include a third container that contains at least one single-stranded oligonucleotide probe complementary to a target nucleic acid and/or a fourth container that contains a hybridization solution and/or a filter sufficient to allow for filtration of aggregated nanoparticles. According to a third embodiment, the kits can include a container that contains the plurality of negatively charged nanoparticles coupled to a substrate. [0020] An eleventh aspect of the present invention relates to a detection device for performing a method of the present invention.
[0021] Assays and kits of the present invention involve the use of negatively charged nanoparticles and nucleic acid molecules, harnessing the electrostatic interactions between the nanoparticles and nucleic acid molecules. In particular, applicants have identified four unique interactions that can be harnessed by the assays and materials of the present invention. These include: (1) the discovery that under certain conditions single stranded nucleic acid will adsorb on negatively charged nanoparticles while double stranded nucleic acid molecules will not; (2) adsorption of single stranded nucleic acid molecules onto the negatively charged nanoparticles suspended in a colloidal solution stabilizes the nanoparticles against salt-induced aggregation; (3) the adsorption rate for single stranded nucleic acid molecules depends on the sequence length; and (4) the adsorption rate for single stranded nucleic acid molecules depends on the temperature of the solution.
[0022] The essential difference between the electrostatic properties of single- stranded and double-stranded nucleic acid probably arises because ss-nucleic acid can uncoil sufficiently to expose its bases while ds-nucleic acid has a stable double helix geometry that always presents the negatively charged phosphate backbone (Watson, The Double Helix: A Personal Account of the Discovery of the Structure of DNA, Weidenfeld and Nicholson, London (1968); Bloomfϊeld et al., Nuclei Acids: Structures, Properties, and Functions, University Science Books, Sausalito, California (1999), each of which is hereby incorporated by reference in its entirety). The negatively charged nanoparticles in solution are typically stabilized by their repulsion, which prevents the strong Van der Waals attraction between the particles from causing them to aggregate (Hunter, Foundations of Colloid Science, Oxford University Press Inc., New York (2001); Shaw, Colloid and Surface Chemistry, Butterworth-Heinemann Ltd., Oxford (1991), each of which is hereby incorporated by reference in its entirety). Repulsion between the charged phosphate backbone of ds-nucleic acid and the negatively charged nanoparticles dominates the electrostatic interaction between the nanoparticle and ds-nucleic acid so that ds-nucleic acid will not adsorb. Because the ss-nucleic acid is sufficiently flexible to partially uncoil its bases, they can be exposed to the negatively charged nanoparticles. Under these conditions, the negative charge on the backbone is sufficiently distant so that attractive Van der Waals forces between the bases and the nanoparticle are sufficient to cause ss-nucleic acid to adsorb to the negatively charged nanoparticle. The same mechanism is not operative with ds-nucleic acid because the duplex structure does not permit the uncoiling needed to expose the bases. In the present invention, the selective adsorption of ss-DNA and RNA to negatively charged nanoparticles (e.g., citrate-coated Au nanoparticles) is documented. In addition, it is shown that adsorption of ss-nucleic acids stabilize the nanoparticles against aggregation at concentrations of salt that would ordinarily screen the repulsive interactions of the negative charge. In the case of metal nanoparticles, their color is determined χ
principally by surface plasmon resonance and this is dramatically affected by aggregation of the nanoparticles (Link et al., Intl. Reviews in Physical Chemistry 19:409-453 (2000); Kreibig et al., Surface Science 156:678-700 (1985); Quinten et al., Surface Science 172:557-577 (1986), each of which is hereby incorporated by reference in its entirety). The difference in the electrostatic properties of ss-nucleic acid and ds-nucleic acid can be used to design a simple colorimetric hybridization assay. The assay can be used for sequence specific detection of untagged oligonucleotides using unmodified commercially available materials. The assay is easy to implement for visual detection at the level of 100 femtomoles, and it is shown that it is easily adapted to detect single base mismatches between probe and target. Also presented herein are initial studies of how length mismatches between target and probe sequence affect the propensity for oligonucleotides to adsorb on metal nanoparticles. [0023] By harnessing the above-identified interactions in the assays and kits of the present invention, the present invention affords methods of detecting target nucleic acids that offer a number of benefits over previously developed detection procedures. Some of these benefits include: no target labeling is required; the assays occur in solution, allowing for detection of the target nucleic acid in less than about 10 minutes (which is significantly faster than chip or surface-based assays that tend to slow down the hybridization process); the detection procedure is temporally separated from the hybridization procedure so that the hybridization process can be optimized with little or no regard to the detection procedure; and the assays can be performed using commercially available materials. The two basic embodiments of the present invention, a colorimetric assay and a fluorimetric assay, afford significant benefits. The colorimetric assay can be performed without the need for expensive detection instrumentation, such as fluorescence microscopes or photomultipliers. Detection of a positive or negative result in the colorimetric assay can be assessed by naked eye of an observer. The assays are extremely sensitive, capable of detecting target nucleic acids in femtomole quantities (or less in the case of the fluorescent approach), capable of discriminating between complex mixtures of nucleic acid, and capable of discriminating between wild-type targets and those bearing SNPs or other mutations such as deletions or modifications such as knockout insertions. Detection of SNPs in genomic DNA is particularly challenging, but is at the forefront of diagnostic technology since it has been associated with a number of hereditary conditions and cancers, and is likely to be responsible for many more (Friedberg, Nature 421 :436- 439 (2003); Futreal et al., Nature 409:850-852 (2002), each of which is hereby incorporated by reference in its entirety).
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 is a pictorial representation of the colorimetric method for differentiating between single and double stranded oligonucleotides; and consequently selective oligonucleotide detection. The circles represent colloidal metal (e.g. gold) nanoparticles.
[0025] Figure 2 is a pictorial representation of the fluorimetric method for selective oligonucleotide detection. The red stars in panels A, B, and D represent identifiable (i.e., unquenched) fluorescence from the fluorescence label on the probe strands. The thin green strands and the thick green strands represent single-strand and double-strand nucleic acid molecules, respectively. The circles in panels C and D represent metal (e.g., gold) nanoparticles. Hybridization between the oligonucleotide probes and target nucleic acid molecules occur before introducing metal nanoparticles. When the nanoparticles are introduced into the hybridization solution where DNA duplex formation did not occur, the fluorescence from the tag on the probe is quenched (panel C). When the nanoparticles are introduced into solution where hybridization occurred, fluorescence from the tag on duplex-forming probes is observed (panel D).
[0026] Figure 3 is a schematic protocol of protein detection combining immuno-PCR with the methods of the present invention. [0027] Figures 4A-B provide evidence for preferential adsorption of ss-DNA on gold nanoparticles. Figure 4A is a graphical illustration of fluorescence emitted from rhodamine red attached to ss-DNA (dashed) and ds-DNA (solid). The fluorescence spectra were recorded from mixtures consisting of the trial hybridization solution (final concentration of the dye labeled ss-DNA: 50 nM), 500 μL of gold colloid, and 500 μL of 10 mM phosphate buffer solution (PBS) containing 0.1 M NaCl. The ss-DNA (dashed) curve was recorded from the mixture containing the probe and its non-complementary target (nc-target). Dot curve was recorded from the W
mixture containing the probe and its complementary target (c-target). Figure 4B is a graphical illustration of Surface Enhanced Resonant Raman Scattering ("SERRS") from Rhodamine Green tagged on ss-DNA (solid) and ds-DNA (dashed). SERRS was recorded from the mixture of 5 picomole probe and 5 picomole nc-target (solid curve) or 5 picomole c-target (dashed curve), and 100 μL of 10 niM PBS containing 0.5 M NaCl, as well as 300 μL silver colloid. The Raman modes at 1645, 1558, 1509, and 1363 cm"1 are aromatic C-C stretching modes of the core of rhodamine green, while the Raman modes at 1279 and 1182 cm"1 are rhodamine C-O-C stretching and C-C stretching vibrations, respectively. [0028] Figures 5A-C show colorimetric detection of oligonucleotide hybridization. Figure 5A is a graph showing absorption spectra of gold colloid (diamonds) and the mixtures containing ss-DNA 1 (circles), ss-DNA2 (triangles), and ds-DNA from the hybridization of ss-DNA 1 and ss-DNA2 (squares), respectively. The gold colloid was diluted with water to the same concentration as in the mixtures. The mixtures contained trial hybridization solution (5 μL (60 μM) ss-DNA in salt buffer solution) added to 500 μL of 17 nM gold colloid, followed by 200 μL of 10 mM PBS and 0.2 M NaCl). Figure 5B is a graphical illustration of the ratio of the absorbance at 520 nm to the absorbance at 700 nm versus oligonucleotide concentration expressed in number of DNA per gold nanoparticle. The DNA sequences and the mixture are the same as in Figure 5 A, except for variation of the amount of DNA. Figure 5C is a photograph showing colorimetric detection of a DNA sequence fragment characteristic of Severe Acute Respiratory Syndrome ("SARS") virus (Drosten et al., The New England Journal of Medicine 348:1967-1976 (2003), which is hereby incorporated by reference in its entirety). All solutions contained 120 picomoles of probe, 200 μL gold colloid, and 100 μL of 10 mM PBS and 0.2 M NaCl. The ratio of the amount of target to the amount of probe in the solutions was 0, 0.2, 0.4, 0.6, and 1 (from left to right), respectively.
[0029] Figures 6A-E show colorimetric detection of targets in mixtures, low concentrations, low amounts, and with single base mismatches. Figure 6A is a photograph showing detection of a target sequence in a mixture. 3.5 μL of trial hybridization solution was mixed with 300 μL of gold colloid and 300 μL of 10 mM phosphate buffer solution containing 0.2 M NaCl. The complementary target contained in the solutions from left to right were 50%, 40%, 30%, and 0% of the total ^
oligonucleotide concentration with non-complementary target making up the remainder. AU solutions contained the 105 picomoles of probe, equal to the total of complementary target and non-complementary target. Figure 6B is a photograph showing detection of target DNA in low concentration solution. 100 μL of gold colloid was diluted in 300 μL water, mixed with 1 μL trial hybridization solution and 300 μL of 10 mM phosphate buffer solution containing 0.3 M NaCl (final target concentration: 4.3 nM). The vial on the left contained unmatched ss-DNA strands while the vial on the right contained complementary strands. Figure 6C is a photograph showing detection of small amounts of target. 5 μL of gold colloid was mixed with 0.2 μL of trial hybridization solutions containing 0.3 μM oligonucleotide then mixed with 3 μL of 10 mM phosphate buffer solution containing 0.2 M NaCl. The resulting droplets of non-complementary ss-DNA mixture (left) and complementary ss-DNA (right) each containing 60 femtomoles were placed on inverted plastic vials for viewing. Figure 6D is a photograph showing identification of single base pair mismatch in ds-DNA via dehybridization kinetics in water. 1 μL of ds-DNA solution dehybridized in 100 μL water for 0, 1, and 2 minutes respectively, then mixed with 300 μl of gold nanoparticles and 300 μL of 10 mM phosphate buffer solution 0.3 M NaCl (final ds-DNA concentration: 0.043 μM). The solution in the left vial of each dehybridization time group contained ds-DNA with a single base pair mismatch while the right vial contained perfectly matched target and probe strands. The red color indicates that part of ds-DNA has dehybridized. Figure 6E is a photograph showing identification of single base pair mismatch in ds-DNA via dehybridization kinetics in gold colloid. 1 μL oligonucleotide and 300 μL of gold nanoparticles were ultrasonicated for 0.5, 1, and 2 minutes, respectively, and then mixed with 300 μL of 10 mM phosphate buffer solution 0.3 M NaCl (final target concentration: 0.05 μM). The solution in the left vial of each dehybridization time group contained ds-DNA with a single base pair mismatch while the right vial contained perfectly matched target and probe strands. The red color indicates that part of ds-DNA has dehybridized. The oligonucleotide sequences are identified in the text.
[0030] Figures 7A-B show that gold nanoparticles preferentially quench the fluorescence from fluorophores labeled on ss-DNA. Figure 7A is a graph showing the fluorescence spectra of the mixtures of 5 μL (10 μM) trial hybridized solution of rhodamine red labeled ss-DNA probe and its complementary target (solid squares), or non-complementary target (open squares), 500 μL of gold colloid and 500 μL of 10 mM PBS containing 0.1 M NaCl. Figure 7B is a graph showing the fluorescence image intensity profile measured with a confocal fluorescence microscope. 0.5 μL (0.1 μM) of the trial hybridization solution was mixed with 500 μL of the diluted gold colloid (diluted with deionized water by factor 20) and 500 μL of 10 mM PBS containing 0.1 M NaCl. Solid circles were recorded from 2 μL of the mixture containing complementary target; open circles from 2 μL of the mixture containing non-complementary target.
[0031] Figures 8A-B show detection of long target and long target in a mixture. Figure 8A is a graph showing the method working with long target. The fluorescence spectra were recorded from the solutions containing complementary target a (solid squares), complementary target b (open squares), and non- complementary target c (solid triangles), respectively. The solution contained 4 μL (10 μM) of trial hybridized solution, 500 μL gold colloid, and 500 μL of 10 mM PBS containing 0. IM NaCl. Figure 8B is a graph showing the method working with long target in a mixture. The fluorescence spectra were recorded from mixtures containing 1% complementary target a (solid squares), 1% complementary target b (open squares), and non-complementary target (solid triangles), respectively. The components of oligonucleotides in the trial hybridized solution contained 10 picomolar non-complementary target, 0.5 picomolar probe, and 0.1 picomolar candidates. The mixtures were made up of 0.5 μL of trial hybridized solutions, 500 μL gold colloid (diluted with 250 μL water), and 500 μL of 10 mM PBS containing 0. IM NaCl.
[0032] Figures 9A-B show single base-pair mismatch detection. Figure 9 A is a graph showing the probe binding in the middle of long target a and target a'. Figure 9B is a graph showing the probe binding at one end of long target b and complementary target b'. The fluorescence spectra for single base-pair mismatch detection were recorded from mixtures containing 1 μL (10 μM) trial hybridized solution (same amount of the probe and the target) warmed in 46°C water bath, 500 μL gold colloid, and 500 μL of 10 mM PBS and 0.1 M NaCl. Solid squares were recorded from the mixtures containing perfect matched ds-DNA and open squares from the mixtures containing ds-DNA with one base-pair mismatch. [0033] Figures 10A-B show simultaneous multiple target detection. Figure
1OA is a graph showing excitation at 570 nm, which is absorption maximum of rhodamine red tagged on probe 1. Figure 1OB is a graph showing excitation at 648 nM, which is absorption maximum of cy5 tagged on probe 2. (Note: The second peak of the spectrum (solid squares) in Figure 1OB is the emission of cy5 tagged on probe 2 excited by 570 nm.) [0034] Figures 1 IA-D show adsorption of ss-DNA to gold nanoparticles. Figure 1 IA graphically illustrates absorption spectra of 300 μL gold colloid and 100 μL deionized water (red), 100 μL of 10 mM PBS (0.2 M NaCl) (blue), 300 picomoles 24 base ss-DNA first, then 100 μL of 10 mM PBS (0.2 M NaCl) (green). Figure 1 IB is a graph showing photoluminescence intensity versus time following addition of 4 picomoles rhodamine red tagged ss-DNAs to 1000 μL gold colloid. 10 mer (red), 24 mer (green) and 50 mer (blue). Figure 11C graphically illustrates absorption spectra of the mixture of 200 picomoles ss-DNA (50 mer) and 300 μL gold nanoparticles heated at different temperature for two minutes, followed by addition of 300 μL of 10 mM PBS (0.2 M NaCl). 220C (blue), 45°C (cyan), 700C (green), and 950C (red). Figure 1 ID graphically illustrates the fluorescence spectra of the hybridized solutions of rhodamine red labeled 15 mer ss-DNA, 50 mer ss-DNA, and gold colloid, the 15 mer binding to 50 mer at middle (red), at end (green) and nowhere (blue). The lower inset schematically illustrates the binding positions between 15 mer and 50 mer. The upper inset contains color photographs of the corresponding mixtures (from left to right) with no fluorescent label on the 15 mer. [0035] Figure 12 is a schematic of the interaction between negatively charged metal nanoparticules and ss-DNA. The wedge-like structure (left) represents the metal nanoparticle, and the structure (right) represents a ss-nucleic acid having a phosphate backbone (solid vertical line) and nucleotide bases (horizontal lines). [0036] Figures 13A-B show identification of PCR amplified DNA sequences. Figure 13A is a schematic of the detection protocol. The mixture of PCR product and probes is denatured and annealed below the melting temperature of the complementary probes, followed by addition of gold colloid. The long blue and green lines represent the PCR amplified DNA fragments and the pink and light blue medium bars the excess PCR primers. The short blue and green bars are complementary probes that bind, resulting in gold nanoparticle aggregation (purple color). The short purple and orange bars are non-complementary probes that do not bind and adsorb to the gold nanoparticles, preventing nanoparticle aggregation and leaving the solution pink. Figure 13B is a color photograph of the resulting solutions with complementary probes (a) and non-complementary probes (b). 8 μL PCR product, 3.5 picomoles probe and 70 μL gold colloid were used in each vial. [0037] Figures 14A-B show single base-pair mismatch detection. Figure 14A illustrates the detection strategy. The red spots on long green and blue lines represent positions of a potential SNP. The long green and blue lines are the complementary sequences of PCR amplified DNA fragment. The short green and blue bars are probes complementary to parts of the wild type sequence of PCR amplified DNA fragment as illustrated. Figure 14B is a photograph showing detection of a single base-pair mismatch. Vials b, d, and f contain PCR product with probes overlapping the single-base mismatch while vials a, c, and e contain PCR product with probes not overlapping the single base pair mismatch. Photographs were taken of the mixtures annealed at 500C (a, b), 54°C (c, d) and 58°C (e, f). 8 μL PCR product, 3.5 picomoles probe and 70 μL gold colloid were used in each vial. [0038] Figures 15 A-B illustrate single base-pair mismatch detection using RNA probes and RNA targets. The symbols shown in Figures 15A-B are as follows: ds: duplex; ds': duplex containing mismatch; ss: control. [0039] Figure 16 illustrates schematically one implementation of the immobilized bead method for separating double stranded from single stranded nucleic acids. Removal of unhybridized short ss-DNA probes by processing the analyte through a filter of packed glass beads (circles filled with grid) functionalized with immobilized negatively charged nanoparticles (shaded circles). The trial hybridization solution prior to the filter is shown schematically above and after the filter below. The fate of the ss-DNA probe (light squiggly line) with tag (open circle resembling sun) is shown on the left, long ss-DNA target in the center and target with hybridized probe on the right. The tag can be fluorescent, radioactive, or electrochemical. The presence of tags in the eluted sample indicates the presence of target. [0040] Figure 17 is a graph showing that ss-DNA is preferentially retained by the column of immobilized beads. [0041] Figure 18 is a graph illustrating the fluorescence of solutions remaining after removal of gold by salt-induced crashout and centrifugation. Solid squares are for a trial analyte that is rhodamine tagged ds-DNA and open squares for a trial analyte with the same amount of rhodamine tagged ss-DNA. [0042] Figures 19A-D illustrate the colorimetric method for RNA sequence detection. In each of Figures 19A-D, the same mixtures of trial hybridization solutions and gold colloid were used. The left vial in each image contains complementary target, the middle vial contains a target with a single base mismatch with the probe, and the right vial contains a random non-complementary target. Each hybridization solution was heated at 94°C for 5 minutes and subsequently annealed at a different temperature for 3 minutes: Figure 19A, 20°C; Figure 19B, 50°C; Figure 19C, 59°C; and Figure 19D, 64°C.
[0043] Figures 20A-B are graphs showing the absorption spectra from the mixtures of trial hybridization solutions annealed at two different temperatures after being added to gold colloid. Squares, circles and triangles from the mixtures contain, respectively, complementary target (c-target), mismatch target (mc-target) and non- complementary target (nc-target). hi each case, the hybridization solutions were heated at 95°C for 3 minutes, then annealed for 1 minute prior to addition to gold colloid at 20°C. DNA Probe: Rhodamine red-5'-AGG AAT TCC ATA GCT-3 ', SEQ ID NO: 8. Wild-type target: 5'-ACU AGG CAC UGU ACG CCA GCUA UG GAA UUC CUU AGC UAU GAG AUC CUU CG-3', SEQ ID NO: 31. Mutant target: 5'- ACU AGG CAC UGU ACG CCA GCUAUG GCA UUC CUU AGC UAU GAG AUC CUU CG-3', SEQ ID NO: 32. [0044] Figure 21 is a graph illustrating detection of single base mutations in RNA sequences using fluorescence quenching of fluorescently labeled DNA probe. Fluorescence spectra of the mixtures of hybridization solution, gold colloid, and buffer/salt solution are illustrated two minutes after mixing. Squares: "Wild-type" RNA target containing a sequence perfectly complementary to the DNA probe. Circles: "Mutant" RNA target containing a sequence forming a single base-pair mismatch with the probe. Mutant probe: Rhodamine red-5 '-AGG AAT TCC ATA GCT-3', SEQ ID NO: 8. Non-complementary background: 5'-CGA UCA CGA GAU CGA-3', SEQ ID NO: 33. [0045] Figure 22 is a graph illustrating the detection of single base mutations in RNA sequences in complex mixtures using the fluorescence assay. P and T denote probe and target, respectively, while w and m indicate wild-type and mutant, respectively. AU hybridization solutions contain non-complementary background RNA at 10 times the concentration of the target. Sequences of the wild-type probe, wild-type target, and mutant target are stated in the description of Figure 21.
DETAILED DESCRIPTION OF THE INVENTION
[0046] The methods of the present invention can be used to detect the presence (or substantial absence) of a target nucleic acid molecule in a sample or test solution. Basically, the method involves combining at least one single-stranded oligonucleotide probe and the test solution under conditions effective to allow formation of a hybridization complex between the at least one single-stranded oligonucleotide probe and any target nucleic acid present in the test solution. If no target nucleic acid or substantially no target nucleic acid is present, then no hybridization complex or substantially no hybridization complex will form. After allowing for hybridization to occur (i.e., if hybridization between the probe and target is possible), the hybridization solution is exposed to a plurality of negatively charged nanoparticles under conditions effective to allow any unhybridized probe to associate electrostatically with the plurality of negatively charged nanoparticles. A determination is then made whether the at least one single-stranded oligonucleotide probe has hybridized to target nucleic acid or electrostatically associated with one or more of the plurality of negatively charged nanoparticles. This determination is made according to an optical property of the hybridization solution, as discussed below. [0047] The methods of the present invention can further include a step for separating ds-nucleic acid from the short single-stranded probe molecules (or other ss- nucleic acids) that remain unbound after the hybridization step, as discussed below. [0048] The target nucleic acid molecule that is intended to be detected can be
DNA or RNA. The DNA or RNA can be isolated directly from samples (i.e., concentrated to be free of cellular debris) and then tested, if present in sufficient quantities, or it can first be amplified by polymerase chain reaction ("PCR") or reverse-transcription PCR. Thus, the DNA to be detected can be amplified cDNA. χ
Because the DNA can be amplified cDNA, the cDNA can also have incorporated therein synthetic, natural, or structurally modified nucleoside bases. [0049] The target nucleic acid molecule can also be from any source organism
(e.g., human or another animal, virus, bacteria, insect, plant, etc.). [0050] As an alternative, the target nucleic acid can contain a nucleotide sequence coupled or otherwise conjugated to a protein or polypeptide. In such case, detection of the target nucleic acid directly confirms presence of the protein or polypeptide. Alternatively, the target nucleic acid can contain a nucleotide sequence coupled or otherwise conjugated to a protein or polypeptide that participates in an immuno-PCR procedure; the subsequently amplified target cDNA confirms indirectly the presence of the target nucleic acid in a sample to be tested (i.e., absence of the target cDNA confirms that the target is not present in the initial sample). [0051] The single-stranded oligonucleotide probes that can be used in the present invention can either be unlabeled or they can be conjugated or otherwise coupled to a label. Suitable labels include, without limitation, fluorescent labels, redox (electrochemical) labels, and radioactive labels.
[0052] Coupling of a fluorescent label to the oligonucleotide probe can be achieved using known nucleic acid-binding chemistry or by physical means, such as through ionic, covalent or other forces well known in the art {see, e.g., Dattagupta et al., Analytical Biochemistry 177:85-89 (1989); Saiki et al., Proc. Natl. Acad. ScL USA 86:6230-6234 (1989); Gravitt et al., J Clin. Micro. 36:3020-3027 (1998), each of which is hereby incorporated by reference in its entirety). Either a terminal base or another base near the terminal base can be bound to the fluorescent label. For example, a terminal nucleotide base of the oligonucleotide probe can be modified to contain a reactive group, such as (without limitation) carboxyl, amino, hydroxyl, thiol, or the like.
[0053] The fluorescent label can be any fluorophore that can be conjugated to a nucleic acid and preferably has a photoluminescent property that can be detected and easily identified with appropriate detection equipment. Exemplary fluorescent labels include, without limitation, fluorescent dyes, semiconductor quantum dots, lanthanide atom-containing complexes, and fluorescent proteins. The fluorophore used in the present invention is characterized by a fluorescent emission maxima that is detectable either visually or using optical detectors of the type known in the art. Fluorophores having fluorescent emission maxima in the visible spectrum are preferred.
[0054] Exemplary dyes include, without limitation, Cy2™, YO-PRO™- 1 ,
YOYO™-1, Calcein, FITC, FluorX™, Alexa™, Rhodamine 110, 5-FAM, Oregon Green™ 500, Oregon Green™ 488, RiboGreen™, Rhodamine Green™, Rhodamine 123, Magnesium Green™, Calcium Green™, TO-PRO™-1, TOTO®-1, JOE, BODIPY® 530/550, DiI, BODIPY® TMR, BODIPY® 558/568, BODIPY® 564/570, Cy3™, Alexa™ 546, TRITC, Magnesium Orange™, Phycoerythrin R&B, Rhodamine Phalloidin, Calcium Orange™, Pyronin Y, Rhodamine B, TAMRA, Rhodamine Red™, Cy3.5™, ROX, Calcium Crimson™, Alexa™ 594, Texas Red®, Nile Red, YO-PRO™-3, YOYO™-3, R-phycocyanin, C-Phycocyanin, TO-PRO™-3, TOTO®-3, DiD DilC(5), Cy5™, Thiadicarbocyanine, and Cy5.5™. Other dyes now known or hereafter developed can similarly be used as long as their excitation and emission characteristics are compatible with a light source and non-interfering with other fluorophores that may be present (i.e., not capable of participating in fluorescence resonant energy transfer or FRET).
[0055] Attachment of dyes to the oligonucleotide probe can be carried out using any of a variety of known techniques allowing, for example, either a terminal base or another base near the terminal base to be bound to the dye. For example, 3'- tetramethylrhodamine (TAMRA) may be attached using commercially available reagents, such as 3'-TAMRA-CPG, according to manufacturer's instructions (Glen Research, Sterling, Virginia). Other exemplary procedures are described in, e.g., Dubertret et al, Nature Biotech. 19:365-370 (2001); Wang et al., J. Am. Chem. Soc, 125:3214-3215 (2003); Bioconjugate Techniques, Hermanson, ed. (Academic Press) (1996), each of which is hereby incorporated by reference in its entirety.
[0056] Exemplary proteins include, without limitation, both naturally occurring and modified (i.e., mutant) green fluorescent proteins (Prasher et al., Gene 111 :229-233 (1992); PCT Application WO 95/07463, each of which is hereby incorporated by reference in its entirety) from various sources such as Aequorea and Renilla; both naturally occurring and modified blue fluorescent proteins (Karatani et al., Photochem. Photobiol. 55(2):293-299 (1992); Lee et al., Methods Enzymol. (Biolumin. Chemilumin.) 57:226-234 (1978); Gast et al., Biochem. Biophys. Res. Commun. 80(l):14-21 (1978), each of which is hereby incorporated by reference in its entirety) from various sources such as Vibrio and Photobacterium; and phycobiliproteins of the type derived from cyanobacteria and eukaryotic algae (Apt et al., J. MoI. Biol. 238:79-96 (1995);. Glazer, Ann. Rev. Microbiol. 36:173-198 (1982); Fairchild et al., J. Biol. Chem. 269:8686-8694 (1994); Pilot et al., Proc. Natl. Acad. ScL USA 81:6983-6987 (1984); Lui et al., Plant Physiol. 103:293-294 (1993); Houmard et al., J. Bacteriol. 170:5512-5521 (1988), each of which is hereby incorporated by reference in its entirety), several of which are commercially available from ProZyme, Inc. (San Leandro, CA). Other fluorescent proteins now known or hereafter developed can similarly be used as long as their excitation and emission characteristics are compatible with the light source and non-interfering with other fluorophores that may be present.
[0057] Attachment of fluorescent proteins to the oligonucleotide probe can be carried out using substantially the same procedures used for tethering dyes to the nucleic acids, see, e.g., Bioconjugate Techniques, Hermanson, ed. (Academic Press) (1996), which is hereby incorporated by reference in its entirety.
[0058] Nanocrystal particles or semiconductor nanocrystals (also known as
Quantum Dot™ particles), whose radii are smaller than the bulk exciton Bohr radius, constitute a class of materials intermediate between molecular and bulk forms of matter. Quantum confinement of both the electron and hole in all three dimensions leads to an increase in the effective band gap of the material with decreasing crystallite size. Consequently, both the optical absorption and emission of semiconductor nanocrystals shift to the blue (higher energies) as the size of the nanocrystals gets smaller. When capped nanocrystal particles of the invention are illuminated with a primary light source, a secondary emission of light occurs at a frequency that corresponds to the band gap of the semiconductor material used in the nanocrystal particles. The band gap is a function of the size of the nanocrystal particle. As a result of the narrow size distribution of the capped nanocrystal particles, the illuminated nanocrystal particles emit light of a narrow spectral range resulting in high purity light. Particles size can be between about 1 nm and about 1000 nm in diameter, preferably between about 2 nm and about 50 nm, more preferably about 5 nm to about 20 nm.
[0059] Fluorescent emissions of the resulting nanocrystal particles can be controlled based on the selection of materials and controlling the size distribution of the particles. For example, ZnSe and ZnS particles exhibit fluorescent emission in the blue or ultraviolet range (-400 nm or less); Au, Ag, CdSe, CdS, and CdTe exhibit fluorescent emission in the visible spectrum (between about 440 and about 700 nm); InAs and GaAs exhibit fluorescent emission in the near infrared range (-1000 nm), and PbS, PbSe, and PbTe exhibit fluorescent emission in the near infrared range (i.e., between about 700-2500 nm). By controlling growth of the nanocrystal particles it is possible to produce particles that will fluoresce at desired wavelengths. As noted above, smaller particles will afford a shift to the blue (higher energies) as compared to larger particles of the same material(s). [0060] Preparation of the nanocrystal particles can be carried out according to known procedures, e.g., Murray et al., MRS Bulletin 26(12):985-991 (2001); Murray et al., IBMJ. Res. Dev. 45(l):47-56 (2001); Sun et al., J. Appl. Phys. 85(8, Pt. 2A): 4325-4330 (1999); Peng et al., J. Am. Chem. Soc. 124(13):3343-3353 (2002); Peng et al., J. Am. Chem. Soc. 124(9):2049-2055 (2002); Qa et al, Nano Lett. l(6):333-337 (2001); Peng et al., Nature 404(6773):59-61 (2000); Talapin et al., J. Am. Chem. Soc. 124(20):5782-5790 (2002); Shevenko et al., Advanced Materials 14(4):287-290 (2002); Talapin et al., Colloids and Surfaces, A: Physiochemical and Engineering Aspects 202(2-3): 145-154 (2002); Talapin et al., Nano Lett. l(4):207-211 (2001), each of which is hereby incorporated by reference in its entirety. Alternatively, nanocrystal particles can be purchased from commercial sources, such as Evident Technologies. [0061] Attachment of a nanocrystal particle to the oligonucleotide probe can be carried out using substantially the same procedures used for tethering dyes thereto. Details on these procedures are described in, e.g., Bioconjugate Techniques, Hermanson, ed. (Academic Press) (1996), which is hereby incorporated by reference in its entirety.
[0062] Exemplary lanthanide atoms include, without limitation, Ce, Pr, Nd,
Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lv. Of these, Nd, Er, and Tb are preferred because they are commonly used in fluorescence applications. Attachment of a lanthanide atom (or a complex containing the lanthanide atom) to the oligonucleotide probe can be carried out using substantially the same procedures used for tethering dyes thereto. Details on these procedures are described in, e.g., Bioconjugate Techniques, Hermanson, ed. (Academic Press) (1996), which is hereby incorporated by reference in its entirety. [0063] When multiple probes are used and each is conjugated to a fluorescent label, it is preferable that the fluorescent labels can be distinguished from one another using appropriate detection equipment. That is, the fluorescent emissions of one fluorescent label should not overlap or interfere with the fluorescent emissions of another fluorescent label being utilized. Likewise, the absorption spectra of any one fluorescent label should not overlap with the emission spectra of another fluorescent label (which may result in fluorescent resonance energy transfer that can mask emissions by the other label). [0064] As noted above, any of a variety of electrochemical or redox labels can be employed. Various electrochemical approaches to DNA detection have been developed (Palecek, E. Talanta 56:809-819 (2002), which is hereby incorporated by reference in its entirety) for detection of oligonucleotide sequences (PCT Application WO 01/42508 to Choong et al; Pividori et al, S. Biosens. Bioelectron. 15:291-303 (2000), each of which is hereby incorporated by reference in its entirety) and DNA damage (Mugweru et al., Anal. Chem. 74:4044-4049 (2002), which is hereby incorporated by reference in its entirety). Electroactivity of the nucleic acids themselves (Mugweru et al., Anal. Chem. 74:4044-4049 (2002); De-los-Santos- Alvarez, Anal. Chem. 74:3342-3347 (2002); Sistare et al., J. Phys. Chem. B 103:10718-10728 (1999); Olivira-Brett et al., Langmuir 18:2326-2330 (2002); Armistead et al., Anal. Chem. 72:3764-3770 (2000); Thorp, Trends in Biotechnol.
16:117-121 (1998), each of which is hereby incorporated by reference in its entirety), incorporation of electroactive markers (PCT Application WO 01/42508 to Choong et al.; Pividori et al., S. Biosens. Bioelectron. 15:291-303 (2000); Yu et al., J. Am. Chem. Soc. 123:11155-11161 (2001); Wang Qt ύ., Anal. Chem. 73:5576-5581 (2001), each of which is hereby incorporated by reference in its entirety) onto the nucleic acids, label-free detection methods using redox reactions modified by DNA hybridization (Ruan et al., Anal. Chem. 74:4814-4820 (2002); Yan et al., Anal. Chem. 73:5272-5280 (2001); Patolsky et al., Langmuir 15:3703-3706 (1999); Patolsky et al., J. Am. Chem. Soc. 123:5194-5205 (2001), each of which is hereby incorporated by reference in its entirety), and selective intercalation of electroactive moieties into duplex DNA have all been demonstrated. A variety of electrochemical measurement protocols have been used including cyclic voltammetry (De-los-Santos-Alvarez, Anal. Chem. 74:3342- 3347 (2002), which is hereby incorporated by reference in its entirety), stripping potentiometry (Wang et al., Anal. Chem. 73:5576-5581 (2001), which is hereby incorporated by reference in its entirety), square wave voltammetry (Mugwera et al., Anal. Chem. 74:4044-4049 (2002), which is hereby incorporated by reference in its entirety), differential voltammetry (Olivira-Brett et al., Langmuir 18:2326-2330 (2002), which is hereby incorporated by reference in its entirety), and AC impedance spectroscopy (Ruan et al., Anal. Chem. 74:4814-4820 (2002); Yan et al., Anal. Chem. 73:5272-5280 (2001); Patolsky et al., Langmuir 15:3703-3706 (1999); Patolsky et al., J. Am. Chem. Soc. 123:5194-5205 (2001), each of which is hereby incorporated by reference in its entirety). Any other suitable electrochemical detection procedure can be employed.
[0065] Exemplary electrochemical labels include, without limitation, a reporter group that contains a transition metal complex (e.g., ruthenium, cobalt, iron, or osmium complexes), or a redox moiety useful against an aqueous saturated calomel reference electrode (e.g., transition metal complexes, 1,4-benzoquinone, ferrocene, ferrocyanide, tetracyanoquinodimethane, N, N, N1, N'-tetramethyl-p- phenylenediamine, or tetrathiafulvalene), and redox moieties useful against an Ag/AgCI reference electrode (e.g., 9-aminoacridine, aciidine orange, aclarubicin, daunomycin, doxorubicin, pirarubicin, ethidium bromide, ethidium monoazide, chlortetracycline, tetracycline, minocycline, Hoechst 33258, Hoechst 33342,7- aminoactinomycin D, Chromomycin A3, mithramycin A, Vinblastine, Rifampicin, Os(bipyridme)-2-(dipyridophenazine)-2"-Co(bipyridine) 331, or Fe-bleomycin). The electrochemical labels can optionally be linked through a suitable linker molecule, typically an organic moiety, as described in PCT Application WO 01/42508 to Choong et al., which is hereby incorporated by reference in its entirety. [0066] The single-stranded oligonucleotide probe can be formed of either
RNA or DNA, and can contain one or more modified bases, one or more modified sugars, one or more modified backbones, or combinations thereof. The modified bases, sugars, or backbones can be used either to enhance the affinity of the probe to a target nucleic acid molecule or to allow for conjugation to a fluorescent label. Exemplary forms of modified bases are known in the art and include, without limitation, alkylated bases, alkynylated bases, thiouridine, and G-clamp (Flanagan et al., Proc. Natl. Acad. ScL USA 30:3513-3518 (1999), which is hereby incorporated by reference in its entirety). Exemplary forms of modified sugars are known in the art and include, without limitation, LNA5 2'-O-methyl, 2'-O-methoxyethyl, and 2'-fluoro (see, e.g., Freier and Attmann, Nucl. Acids Res. 25:4429-4443 (1997), which is hereby incorporated by reference in its entirety). Exemplary forms of modified backbones are known in the art and include, without limitation, phosphoramidates, thiophosphoramidates, and alkylphosphonates. Other modified bases, sugars, and/or backbones can, of course, be utilized.
[0067] The single-stranded oligonucleotide probes can be of any length that is suitable to allow for rapid hybridization to target nucleic acids (if present) in the test solution, and rapid electrostatic association with negatively charged nanoparticles later introduced into the test solution. By rapid, it is intended that the single-stranded oligonucleotide probe can electrostatically associate with negatively charged nanoparticles at a rate that is greater (preferably by at least an order of magnitude) than the rate of association with other nucleic acids in the test solution prior to introduction of the oligonucleotide probe. By way of example and without limitation, the single-stranded oligonucleotide probes are preferably between about 10 and about 50 nucleotides in length, more preferably between about 10 and 30 nucleotides in length, most preferably between about 12 and 20 nucleotides in length. [0068] The single-stranded oligonucleotide probes can have their entire length or any portion thereof targeted to hybridize to the target nucleic acid. It is preferable for the oligonucleotide probe to have a nucleotide sequence that is 100 percent or perfectly complementary to part of the target nucleic acid sequence. [0069] The amount of oligonucleotide probe introduced into the test solution can be determined based upon the total amount of negatively charged nanoparticles to be introduced into the hybridization solution and/or the total amount of target nucleic acid that is believed to be present.
[0070] For the colorimetric assay (described below), it is preferable that the amount of oligonucleotide probe is at least slightly greater than the amount of negatively charged nanoparticles present in the hybridization solution (i.e., greater than a 1 : 1 ratio), more preferably greater than about 10:1, and up to about 30:1. A reasonable match in the amounts of probe and target used are desirable for optimization of the assay. If the amount of nucleic acid in a sample can be reasonable estimated, then the ratio of probe:target should be between about 0.3:1 and about 3:1. If reasonable estimates cannot be made, then concentration series can be performed. [0071] For the fluorescent assay described below, the relative concentrations of target and probe in the trial solution are not critical. Instead, an excess of negatively charged nanoparticles is utilized so that all the unhybridized probes will be quenched (and excess target does not produce fluorescence). [0072] For the electrochemical or radiation assays described below, an excess of negatively charged nanoparticles is also used so that all unhybridized probes can be aggregated for separation of the bound and unbound nucleic acids. [0073] When more than one single-stranded oligonucleotide probe is utilized at a time, the same criteria disclosed above can be taken into consideration. [0074] The oligonucleotide probe can be synthesized using standard synthesis procedures or ordered from commercial vendors, such as Midland Certified Reagent Co. (Midland, Texas) and Integrated DNA Technologies, Inc. (Coralville, Iowa). The commercially ordered probes can be obtained with the desired label. [0075] The negatively charged nanoparticles can be formed of either a conductive metal or an uncharged substrate, such as glass.
[0076] The metal nanoparticles can be formed of any conductive metal or metal alloy that allows the nanoparticle to be capable of electrostatically associating with a single-stranded nucleic acid molecule or aggregating with other metal nanoparticles under appropriate conditions. (Prior to use in the present invention, it should be appreciated that the colloidal suspension maintains the metal nanoparticles in a stable environment in which they are substantially free of aggregation.) Importantly, the metal nanoparticles do not significantly associate electrostatically with hybridization complexes (that is, double-stranded nucleic acid molecules). Exemplary metal nanoparticles include, without limitation, gold nanoparticles, silver nanoparticles, platinum nanoparticles, mixed metal nanoparticles (e.g., gold shell surrounding a silver core), and combinations thereof. In some embodiments, the metal nanoparticles can be magnetic, formed of a magnetic inner core such as cobalt and an outer core such as gold. [0077] Suspensions of colloidal metal nanoparticles can be formed using the procedures described in Grabar et al., Anal. Chem. 67:735-743 (1995), which is hereby incorporated by reference in its entirety. The metal nanoparticles in certain embodiments do not contain any ligands conjugated or otherwise bound to their outer surface. They are, however, stabilized in the solution by negatively charged anions, such as those identified in the paragraph below. The colloidal suspension preferably contains metal nanoparticles of between about 5 nm and about 500 nm, most preferably between about 10 nm and 30 nm.
[0078] The nanoparticle formed of an uncharged substrate is preferably charged using anions or polyanions. The anions or polyanions can be coupled to the substrate (e.g., glass) using standard glass binding chemistry. Exemplary anions include, without limitation, citrate, acetate, carbonate, dihydrogen phosphate, oxalate, sulfate, and nitrate. Exemplary polyanions include, without limitation, poly(2- acrylamido-2-methyl-l-propanesulfonic acid), poly(acrylic acid), poly(anetholesulfonic acid), poly(anilinesulfonic acid), poly(sodium 4- styrenesulfonate), poly(4-styrenesulfonic acid), and poly(vinylsulfonic acid). Other anions and polyanions can also be employed.
[0079] In practicing the assay, the detection of hybridization between probe and target can be achieved in one of several preferred approaches: a colorimetric approach, a fluorimetric approach, and a redox or radiation approach. Each has a distinct advantage over the other and can be employed as desired. [0080] In a colorimetric assay (in which the probe can be unlabeled), the optical property of the hybridization solution is the visible color thereof. In this embodiment, the negatively charged nanoparticles are preferably the metal nanoparticles. A color change of the hybridization solution can be brought about by inducing aggregation of the plurality of metal nanoparticles as illustrated in Figure 1. The colorimetric assay is particularly useful when quantification is not necessary and where expensive detection equipment is unavailable. Detection of the color change in the hybridization solution can be carried out by naked eye observation of a user (i.e., the person performing the assay).
[0081] Aggregation will only occur if an insubstantial number of oligonucleotide probes has electrostatically associated with the metal nanoparticles. If a substantial number of oligonucleotide probes has electrostatically associated with the metal nanoparticles (on average greater than about one or two per nanoparticle), aggregation will be inhibited noticeably. Aggregation (color change) indicates that the target nucleic acid was present in the test solution. Induction of aggregation can be carried out by introducing a salt solution into the hybridization solution, with the salt being of sufficient concentration to alter the electrostatic properties of the metal nanoparticles, thereby promoting their aggregation. The salt solution preferably comprises a Na+ concentration of between about 0.01 and about 1 M, more preferably between about 0.1 and about 0.3 M. The introduction of the salt solution to the hybridization medium can either be carried out simultaneously with the introduction of the solution containing the metal nanoparticles, or in succession therewith (either with or without a delay of up to about 15 minutes).
[0082] Because the colorimetric assay can be detected by naked eye observation, a user can either examine the hybridization solution for a detectable change in color or the assay can be carried out in parallel with one or more controls (positive or negative) that replicate the color of a comparable solution containing aggregated metal nanoparticles (negative control) and/or a comparable solution containing substantially non-aggregated metal nanoparticles (positive control). [0083] In the fluorimetric assay, the optical property of the hybridization solution is the fluorescence spectrum or the magnitude of a fluorescence peak by a fϊuorophore. The photoluminescent property of the fluorophore label is detected after the hybridization procedure is allowed to proceed in the presence of the negatively charged nanoparticles. Non-hybridizing oligonucleotide probes, based on their size, will more rapidly associate electrostatically with the negatively charged nanoparticles than longer nucleic acid molecules in the hybridization solution. Depending upon the type of negatively charged nanoparticles being employed, the aggregates may or may not need to be separated from the nanoparticles remaining in solution. [0084] With use of the metal nanoparticles, separation is not required because the absence of hybridization (i.e., absence of the target) is indicated by substantial quenching of fluorescence by the fluorescent label when oligonucleotide probes electrostatically associate with one or more metal nanoparticles. Hybridization between the oligonucleotide and the target nucleic acid molecule (i.e., presence of the target) is indicated by a maintained photoluminescent property even after aggregation of the metal nanoparticles (which is achieved in the same manner as described above). These alternatives are illustrated in Figure 2. [0085] With the use of non-metallic nanoparticles that do not necessarily quench fluorescent emissions, labeled probes remaining in solution (i.e., in a ds- hybridization complex) are physically separated from aggregates (to which ss-nucleic acids and probes have bound). Fluorescent emissions from the eluent (solution) -∑y-
indicate presence of the ds-nucleic acid and, hence, the target. Any of a variety of physical separation procedures can be employed, as described infra. [0086] The fluorimetric assay is particularly useful for high sensitivity, when the target of interest is only one or many nucleic acid strands in a sample, when quantification of the target nucleic acid is desired, or when the presence of multiple distinct target nucleic acid molecules are being simultaneously analyzed within the same hybridization solution (i.e., using multiple oligonucleotide probes each with a distinct fluorophore attached thereto). Detection of the fluorescence properties of the hybridization solution can be achieved using appropriate detection equipment as is known in the art (e.g., fluorescence microscope, photomultipliers, CCD cameras, photodiodes, etc.).
[0087] Because the fluorimetric assay involves measuring fluorescence caused by the fluorophore(s) in the hybridization solution, a user can either examine the hybridization solution for the presence or absence of fluorescence. No controls are necessary.
[0088] Because the fluorimetric assay is highly sensitive to even small quantities and the photoluminescent properties can be detected with precise instrumentation, the fluorimetric assay lends itself to quantifying the amount of a target nucleic acid present in a test solution. One approach for quantifying the amount of target nucleic acid present in the test solution involved comparing the results from the test solution to the results obtained from two control solutions that each contain known but differing amounts of the target nucleic acid. Thus, measurements of the photoluminescent property are obtained from the test solution and the two control solutions. Based on the photoluminescence of each solution, it is possible to calculate the quantity of the target nucleic acid in the test solution relative to the quantity of the target nucleic acid present in the first and second control solutions. Alternatively, the quantity of the target nucleic acid in the test solution can be calculated using the measured optical property (from the test solution) and a calibration curve of measured optical (e.g., photoluminescence) properties versus quantity of target nucleic acid.
[0089] From the foregoing description of the fluorimetric assay, it should be appreciated that, in principle, fluorescence is extremely sensitive. In fact, from personal experience the applicants have demonstrated in other work that single 3o
molecule fluorescence can be achieved, allowing for detection of single copies of DNA. This can effectively obviate the need for PCR amplification altogether. [0090] The improvement described below resolves two limitations of the fiuorimetric assay described above. The first limitation involves the contrast between unquenched fluorescence and fluorescence of hybridized probe. This can arise when the target to be hybridized represents a small enough fraction of the sample that it is overwhelmed by probe fluorescence that is not completely quenched. This can also arise if there were trace luminescence from the gold particles themselves. The second limitation is that single (or very few) molecule sensitivity can be achieved when it is known that the fluorescent molecule is within a very limited area. Hence, to exploit the theoretical sensitivity of the fiuorimetric assays, it would not be enough to improve the contrast alone. The hybridized DNA with the fluorescent probe should be localized so that fluorescence can be collected by a fluorescence microscope or, better still, a confocal microscope. Both of these detection schemes are able to afford visual detection of single molecules, because the area from which they collect light is so small that the stray background becomes negligible compared to the probe. [0091] Thus, an improvement of the present invention relates to overcoming the limits of sensitivity of the fluorimetric assay described above. Following hybridization and prior to detection, the product of the hybridization procedure (which contains unbound ss-probe, ss-nontarget nucleic acid, and ds-target nucleic acid) is treated to allow for separation of the ds-target nucleic acid from the unbound ss-probe and ss-nontarget nucleic acid.
[0092] Exemplary approaches for treating the hybridization product for separating the ds-target nucleic acid include, without limitation: (1) the use of immobilized, electrostatically charged nanoparticles (e.g., citrate-coated gold or polyanion-coated glass); (2) causing electrostatically charged nanoparticles, with ss- nucleic acids bound thereto, to form insoluble aggregates (the so-called "crashout" approach); (3) concentrating ds-nucleic acid onto a charged solid surface (which can be performed alone or in combination with either of (1) or (2)); (4) the use of magnetic, electrostatically charged nanoparticles, which can be removed from solution with ss-nucleic acid adsorbed thereto; (5) the use of surfaces functionalized with thiol moieties to remove gold from solution; (6) addition of soluble dithiol or thioamine compounds to react with gold nanoparticles and remove them from solution via aggregation; or (7) mechanical methods to filter and remove the nanoparticles, such as centrifugation or passing the solution through a nanoporous network capable of removing the aggregated nanoparticles while allowing ds-nucleic acid hybridization complexes to pass through with the adsorbed tagged probes (for example, pushing the solution through a nylon membrane).
[0093] A modified approach for aggregation and separation involves the use of functionalized gold nanoparticles. For example, relatively large gold nanoparticles (about 30 run up to about 100 nm, preferably about 40 to about 60 nm), whose surface is modified with mixed thiol self-assembled layers, can be used. Most of the surface can contain HS-(CH2)nCOOH to make the particle nominally water soluble and negatively charged so as not to adsorb ds-DNA. A few sites per particle can be thiolated with HS-(CH2)mSH dithiols that would allow for attachment to other gold nanoparticles, thereby forming aggregates that would crash out the gold/ss-DNA. It is preferably for m>n to facilitate the process. Though slow, the process should be effective in aggregating the gold nanoparticles.
[0094] Thiolated surfaces can also be prepared using of a variety of glass surfaces, e.g., a column of glass beads with an exposed thiol can be fabricated using standard silanization chemistry. [0095] As an alternative to the fmorimetric labeling of probes, non-fluorescent labeling can be utilized, such as electrochemical or radioactive labeling using known (or hereafter developed) electrochemical or radioactive labels. These detection procedures can be used with separation, and preferably also with concentration of the ds-nucleic acid (carrying the probe). Electrochemical and radiation detection procedures are known in the art and can easily be adapted for detection of the labels, especially following separation protocols described above.
[0096] One of the important uses of the assays of the present invention is with one or more forms of PCR, as noted above. Because PCR can quickly amplify the total amount of nucleic acid in a sample, it is often used with hybridization-based detection procedures. One of the significant benefits of the present invention is that the assay can be performed using the hybridization medium employed in the thermocycler. The only requirement, however, is that the product of PCR (typically a double-stranded cDNA) must be denatured prior to introducing the negatively charged nanoparticles. Specifically, the double-stranded cDNA can be denatured before or ^
after introducing the oligonucleotide probe to the hybridization medium, but before introducing the negatively charged nanoparticles. Failure to denature the double- stranded cDNA will preclude hybridization between any target nucleic acid, if present, and the oligonucleotide probe, resulting in a possibly false negative result. Alternative PCR procedures that achieve a single-stranded product can be used without denaturing the PCR product.
[0097] Another important use of the assays of the present invention is for detecting a single nucleotide polymorphism ("SNP") in a target nucleic acid molecule. This is performed in slightly different manners depending on whether the colorimetric assay or the fluorimetric (or electrochemical or radiation) assay is to be performed. [0098] Basically, the colorimetric assay is performed in parallel using a test solution and a control solution. The test hybridization solution contains a target nucleic acid molecule and at least one first single-stranded oligonucleotide probe having a nucleotide sequence that hybridizes to a region of the target nucleic acid molecule that may contain a SNP. The probe contains a nucleotide sequence that does not hybridize perfectly to the region containing the SNP (i.e., no base-pairing occurs with the SNP). The control hybridization solution contains the target nucleic acid molecule and at least one second single-stranded oligonucleotide probe including a nucleotide sequence that hybridizes perfectly to a region of the target nucleic acid molecule that does not contain a single-nucleotide polymorphism. Both the test and control hybridization solutions are then exposed to the metal nanoparticles, allowing any unhybridized probes in the hybridization solutions to electrostatically associate with the metal nanoparticles. Importantly, during this stage of the assay, the hybridization solutions are maintained at a temperature that is between the melting temperature of the at least one first single-stranded oligonucleotide probe and the melting temperature of the at least one second single-stranded oligonucleotide probe (which has a higher melting temperature because it is perfectly complementary). Depending on the assay being performed (colorimetric or fluorimetric ), a determination is made whether an optical property of the test and control hybridization solutions are substantially different. A substantial difference indicates the presence of the single nucleotide polymorphism in the target nucleic acid molecule. [0099] In detecting SNPs, the first and second single-stranded oligonucleotide probes can possess the same nucleotide sequence (and be the same length) or a different nucleotide sequence. That is, the two oligonucleotide probes can hybridize to the same region of the target nucleic acids or different regions. If the latter, then the target nucleic acid molecule in the control solution is, e.g., a cDNA molecule that is known not to possess the particular SNP being detected in the test solution. If the former, then the hybridization region of the target nucleic acid molecule in the control solution is known to be stable and free of SNPs (i.e., contains a wild-type sequence). To enhance the difference between the melting temperatures of the two oligonucleotide probes with their respective targets, the oligonucleotide probe for the control assay can be longer or can possess a modified structure (e.g., modified bases, backbone, etc.) that enhances the stability between the probe and target. [0100] The fluorimetric assay is performed substantially as described above, except that the temperature of the hybridization solution is measured when quenching of photoluminescence from the fluorescent label begins (i.e., the temperature is slowly reduced until quenching begins). The measured temperature represents the melting temperature between the probe and the target nucleic acid. This measured melting temperature is then compared to a known melting temperature of a perfectly complementary probe (this measurement can either be provided with a commercial kit or measured by performing the assay in parallel). A difference between the melting temperatures indicates the presence of the single nucleotide polymorphism in the target nucleic acid molecule. These assays can also be performed when using the separation and detection procedures described above. [0101] Yet another important use of the assays of the present invention is for detecting the presence of a pathogen in a sample. Basically, a sample is obtained
(e.g., tissue sample, food sample, water sample, etc.) and nucleic acid is isolated from the sample. Having isolated the nucleic acid, either RNA or DNA, an assessment can be made as to whether enough of the sample is present to afford detection using the assays or whether PCR or RT-PCR is necessary to amplify the isolated nucleic acid. Thus, amplification may or may not be necessary. For example, total RNA isolated from a sample may be of sufficient quantity to proceed without RT-PCR; whereas total DNA isolated from a sample may require amplification. Regardless, the assay of the present invention is performed and the optical property (color or fluorescence intensity) of the hybridization solution is measured or assessed to determine whether or not the single-stranded oligonucleotide probe has hybridized to the target nucleic acid, indicating presence of the pathogen. This assay can also be performed when using the separation and detection procedures described above. [0102] Yet another important use for the assays of the present invention is for genetic screening. Basically, a sample is obtained from a patient and nucleic acid is isolated from the sample. Because genetic screening will typically involve DNA isolation and analysis, it will typically (though not necessarily) require amplification. Regardless, the assay of the present invention is performed and the photoluminescent property of the hybridization solution is measured or assessed to determine whether or not the single-stranded oligonucleotide probe has hybridized to the target nucleic acid, indicating presence of a genetic marker for a genetic condition, a hereditary condition (e.g., paternity, maternity, relatedness, etc.), or identifying an organism. This assay can also be performed when using the separation and detection procedures described above.
[0103] A further use of the assays of the present invention is detection of a protein or antibody in a sample. Immuno-PCR is a procedure that can afford cDNA amplification only if a targeted protein is present in a sample. Thus, the assays of the present invention can be coupled with the amplification detection procedure of immuno-PCR to confirm presence of the amplified cDNA in the hybridization medium and, thus, the target protein in a sample. Basically, a sample is obtained and immuno-PCR is performed using the sample, wherein the immuno-PCR results in amplification of a nucleic acid that is conjugated to a protein. Thereafter, the assays of the present invention are performed where the nucleic acid that is conjugated to the protein (or its complement) becomes the target of the colorimetric or fluorimetric assay of the present invention. This assay can also be performed when using the separation and detection procedures described above.
[0104] A further use of the assays of the present invention is quantifying the amount of amplified nucleic acid prepared by polymerase chain reaction (or similar amplified procedure). Basically, one or more, and preferably two or more fluorescently labeled oligonucleotide primers are provided that each have a nucleotide sequence capable of hybridizing to a nucleic acid molecule, or its complement, that us to be amplified. Amplification using the primers is carried out using any of a variety of known amplification procedures (such as polymerase chain reaction) using a target nucleic acid molecule, and/or its complement, and the provided fluorescently labeled oligonucleotide primers. Thereafter, the fluorimetric method of the present invention is performed on a sample obtained after the amplification procedure has been performed. The level of fluorescence detected from the sample indicates the amount of primer that has been incorporated into an amplified nucleic acid molecule. As amplification continues (and incorporated more of the primers into longer, amplified sequences), the amount of fluorescence from a given sample should increase due to the reduced rate at which longer nucleic acid electrostatically associate to the metal nanoparticles. Unextended primers, on the other hand, will rapidly associate with the metal nanoparticles, which results in quenching of fluorescence by labels attached thereto. This assay can also be performed when using the separation and detection procedures described above. [0105] A further aspect of the present invention relates to one or more types of kits that can be used to practice the assays of the present invention. The kits can include, among other components, various containers that contain individual components that are used in accordance with the methods of the present invention, as well as instructions for carrying out one or more embodiments of the invention. [0106] According to one embodiment, the kit includes a first container that contains a colloidal solution of metal nanoparticles, and a second container that contains an aqueous solution containing at least one single-stranded oligonucleotide probe having a nucleotide sequence that is substantially complementary to a target nucleic acid molecule. Depending on the assay to be performed (colorimetric or fluorimetric ), the oligonucleotide probe in the second container may or may not be conjugated to a fluorescent label of the types described above. With fluorimetric assays and the ability to discriminate between multiple targets, the second container can optionally contain additional oligonucleotide probes (directed to the same or different target nucleic acid molecules), each having a distinct fluorescent emission pattern. In addition to the foregoing containers and components, containers containing control solutions, salt solutions, and various instructions can also be provided.
[0107] According to another embodiment, the kit includes a first container that contains a colloidal solution of negatively charged nanoparticles, and a second container that contains an aqueous salt solution suitable to induce aggregation of the negatively charged nanoparticles. This particular kit format is desired when the user intends to supply their own probe (with labels) and detection equipment. That is, depending upon the probes employed, detection devices suitable for electrochemical labels, radioactive labels, or fluorescence labels can be employed as desired. The kit can optionally include a filter that is suitable to remove salt-induced aggregates while allowing passage of non-aggregated nanoparticles and ds-nucleic acids, as well as instructions for performing the assays of the present invention. [0108] According to a further embodiment, the kit includes a plurality of negatively charged nanoparticles bound to a substrate, for example, glass beads. The substrate can be packed into a column, where they act as a filter to remove short, ss- nucleic acid while allowing ds-nucleic acid to flow through. The kit can also include instructions for performing the assays of the present invention.
EXAMPLES
[0109] The following examples are provided to illustrate embodiments of the present invention but they are by no means intended to limit its scope.
Materials and Methods for Example 1 [0110] A colloidal solution of gold nanoparticles of about 13 nm diameter synthesized via citrate reduction OfHAuCl4 (Grabar et al., Anal. Chem. 67:735-743 (1995), which is hereby incorporated by reference in its entirety) was used. The concentration of the colloidal solution was typically 17 nM. Lyophilized oligonucleotide sequences and their complements were purchased from MWG Biotech (High Point, NC) and dissolved in 10 mM phosphate buffer solution.
Typically, attempted hybridization of the probe and the target was conducted at room temperature for 5 minutes in 10 mM phosphate buffer solution containing 0.3 M NaCl. Specific salt concentrations vary with experiment and are stated in the figure captions. Following the trial hybridization, the trial solution was mixed with gold colloid and immediately followed by addition of salt/buffer solution.
[0111] Samples were placed in quartz cuvettes with 5 mm path length to record absorption spectra using a Perkin Elmer UV/VIS/NIR spectrometer Lambda 19 with water as a reference. For fluorescence spectra and intensities versus time, dye ^
labeled oligonucleotides purchased from MWG Biotech (High Point, NC) were used. Solutions in quartz cells with 1 cm path length were studied on a Jobin-Yvon Fluorolog-3 spectrometer with front face collection geometry and 4 nm resolution. Resonance Raman spectra were taken on these dye labeled oligonucleotides with steady state 532 nm excitation and detection by an Ocean Optics CCD array with a holographic notch filter to reject Rayleigh scattering. The resolution was approximately 10 cm"1. Photographs were taken with a Canon S-30 digital camera.
Example 1 - Gold Nanoparticles Preferentially Adsorb Single-Stranded Nucleic Acid Rather Than Double-Stranded Nucleic Acid
[0112] Direct evidence for the preferential interaction between dye-tagged ss-
DNA and gold nanoparticles is illustrated in Figures 4A-B. The fact that dye-tagged ss-DNA adsorbs on the gold while ds-DNA does not can be seen through the effects of adding colloidal gold to solutions containing either dye-tagged ss-DNA or dye- tagged ds-DNA. In the case of dye tagged ss-DNA, quenching of the dye photoluminescence and enhancement of resonant Raman scattering from the dye were observed. Both of these require intimate contact between the dye and the gold since they are effects of electronic interactions with the gold plasmons. [0113] Figure 5 A presents spectra of the colloid prior to and after addition of ss-DNA or ds-DNA and salt/buffer solution. Ordinarily, exposure to salt screens the repulsive interactions and causes colloid aggregation (Hunter, Foundations of Colloid Science, Oxford University Press Inc., New York (2001); Shaw, Colloid and Surface Chemistry, Butterworth-Heinemann Ltd., Oxford (1991), which are hereby incorporated by reference in their entirety). Apparently, the adsorption of the ss-DNA based on the gold nanoparticles additionally stabilizes the colloidal gold particles against aggregation when salt is introduced. Thus, solutions with adequate quantities of ss-DNA prevent aggregation and the gold colloid remains pink while solutions with ds-DNA do not affect the aggregation and the solutions turn blue. Presumably, this has to do with a redistribution of charge that makes the surface appear more negatively charged. The Raman studies suggest that the ss-DNA does not replace the citrate ions.
[0114] Figure 5B illustrates a condensed form of the same data for two ss-
DNA sequences and documents how the color depends on the amount of ss-DNA. ,o
-38-
Remarkably, solutions with only a few ss-DNA per gold nanoparticle have distinctly different absorption spectra in spite of the fact that the surface area of the nanoparticles is sufficient to accommodate several hundred ss-DNA 24-mers. With enough ss-DNA, the colloid retains a pink coloring while hybridization of the trial solution to form ds-DNA leads to a bluish colloid (Figure 5C). From a practical point of view, this allows the design of an assay to determine whether a given sample contains single stranded or double stranded DNA along the lines of the protocol depicted in Figure 1. An extremely important feature of the method is that hybridization can be done with label free oligonucleotides under optimized conditions (pH, salt, and buffer concentrations) and is completely independent of the detection step. Also investigated is what happens with concentration mismatches between target and probe by using solutions where their ratio is varied from 0 to 1. The results (Figure 5C) prove the technique to be surprisingly robust in its ability to detect the presence of the target. Calibrated colorimetric measurements could be used to determine the amount of target quantitatively.
[0115] Similarly, one can consider the case where the analyte solution contains a mixture of oligonucleotide sequences as might occur in products of polymerase chain amplification, where primers and other fragments are present (Rolfs et al., PCR: Clinical Diagnostics and Research, Springer- Verlag, Berlin Heidelberg (1992), which is hereby incorporated by reference in its entirety). Figure 6 A illustrates the result for a mixed oligonucleotide analyte with various fractions of target sequence and it is clear that as little as 30% target is easily detected. A situation similar to concentration mismatch occurs when the target and probe sequences are complementary but have different lengths. In that case, one could imagine that some of the hybridized chain appears to have the electrostatic properties of ss-DNA while other portions appear double stranded. Qualitatively, the results are similar to those with perfect length match and even hybridized probe and target strands with relatively large length differences (on the order of 5-10 base pairs) behave as double stranded. [0116] The extraordinarily high extinction coefficient of gold nanoparticles
(Doremus, J. Chem. Phys. 40:2389-2396 (1994), which is hereby incorporated by reference in its entirety) makes the colorimetric method extremely sensitive. At 17 nM concentration (Grabar et al., Anal. Chem. 67:735-7 '43 (1995), which is hereby ^
incorporated by reference in its entirety), a 1 cm path length provides optical densities near unity. Empirically, it is easy to visually identify the colour in 5 μL droplets that contain less than 100 femtomoles of gold particles. Figure 5B illustrates that ss-DNA concentrations only slightly greater than the nanoparticle concentration are sufficient to stabilize the colloid against aggregation when exposed to salt. Consequently, one would expect to be able to differentiate between amounts of ss- and ds-DNA of order 100 femtomoles without instrumentation. Even though adsorption of only one or two ss-DNA strands per nanoparticle covers very little of the gold's surface area, it appears to add net negative charges that are distributed around the nanoparticle through rearrangement of charges in the citrate coating. Consistent with the above reasoning, target concentrations of 4.3 nM (Figure 6B) or total amounts of target as low as 60 femtomoles (Figure 6C) produce easily visible differences. Utilizing an absorption spectrometer to evaluate color should produce at least an order of magnitude improvement in sensitivity and use of a null method for measuring absorption, such as photo-thermal deflection, would still further enhance sensitivity (Jackson, Applied Optics 20:1333-1344 (1981), which is hereby incorporated by reference in its entirety).
[0117] The method is easily adapted to identifying single base pair mismatches between probe and target as is essential for detection of biologically important single nucleotide polymorphisms (Rolfs et al., PCR: Clinical Diagnostics and Research, Springer- Verlag, Berlin Heidelberg (1992), which is hereby incorporated by reference in its entirety). Utilized was the fact that the kinetics of ds- DNA dissociation into ss-DNA fragments depend on the binding strength (Owczarzy et al, Biopofymers 44:217-239 (1997); Santalucia et al., J. Am. Chem. Soc. 113:4313- 4322 (1991), which are hereby incorporated by reference in their entirety) and are therefore faster for mismatched ds-DNA (ds'-DNA) than for perfectly matched ds- DNA. The ds-DNA from the trial solution was allowed to dehybridize briefly in water without salt before adding gold colloid and the salt/buffer solution. An obvious color difference was observed between perfectly matched (5'-TAC GAG TTG AGA ATC CTG AAT GCG-3 ' (SEQ ID NO: 1) and its complement) and single base pair mismatched ds-DNA segments SEQ ID NO: 1 and 5'-CGC ATT CAG GCT TCT CAA CTC GTA-3' (SEQ ID NO: 3) waiting 2 minutes before performing the assay (Figure 6D). While dehybridization can also be done in the gold colloid solution simply by delaying the introduction of the buffer/salt solution, the ds-DNA is found more stable in the colloid solution than in water, and there is no significant dehybridization as determined by the assay after 10 minutes in gold colloid. A single base pair mismatched DNA segment showed obvious dehybridization after 5 minutes. Subjecting the mixture of oligonucleotide solution and gold colloid to ultrasound for 1 or 2 minutes before mixing with buffer/salt solution accelerated the dehybridization and also gave excellent contrast between ds-DNA and ds'-DNA (Figure 6E). [0118] It has been demonstrated that ss-DNA and ds-DNA have different propensities to adsorb on gold nanoparticles due to their electrostatic properties. This has been used to design an oligonucleotide recognition assay that uses only commercially available materials, takes less than ten minutes, requires no detection apparatus, is sensitive to single base mismatches, and is reasonably tolerant of concentration or length mismatches. The assay described has additional benefits beyond its speed and simplicity. Because of the ability to exploit the electrostatic properties of the DNA, hybridization is separated from detection so that the kinetics and thermodynamics of DNA binding are unperturbed by steric constraints associated with probe functionalized surfaces. In addition, the assay is homogeneous as it occurs exclusively in the liquid phase, a feature that makes it easy to automate using standard robotic manipulation of microwell plates. The ability to differentially adsorb ss-DNA onto the gold particles can also form the basis for a sensitive assay based on fluorescence that still avoids tagging of the analyte. With fluorescent dyes incorporated onto the probe strands, the fluorescence of the ss-DNA can be selectively quenched as in Figure 4A since it forces the dye to be near the gold nanoparticles where the fluorescence is quenched (Dubertret et al., Nature Biotechnol. 19:365-370 (2001); Du et al., J. Am. Chem. Soc. 125:4012-4013 (2003), which are hereby incorporated by reference in their entirety). If the tagged probe ss-DNA binds the target, however, the ds-DNA does not adsorb on the gold and the fluorescence persists. [0119] Surface plasmon resonance imbues isolated 13 nm diameter Au-nps with a sharp absorption ~ 520 nm and a corresponding reddish hue (Kreibig and Genzel, "Optical Absorption of Small Metallic Particles," Surf. Sd. 156:678-700 (1985), which is hereby incorporated by reference in its entirety). Aggregation of these Au-nps leads to interparticle plasmon interactions that substantially change the spectrum to a very broad absorption throughout the visible and a corresponding grayish-blue color (Quinten and Kreibig, "Optical Properties of Aggregates of Small Metal Particles," Surf. Sd. 172:557-577 (1986), which is hereby incorporated by reference in its entirety). Colloidal Au-np suspensions are stabilized against Au-np aggregation by adsorption of negatively charged ions that lead to strong electrostatic repulsion between the nanoparticles (Hunter, Foundations of Colloid Science. Oxford University Press Inc., NY (2001), which is hereby incorporated by reference in its entirety). Most commonly, sodium citrate is added to gold nanoparticles during their synthesis so that citrate adsorption makes the Au-np surfaces negatively charged. Both the colorimetric and fluorescent detection protocols take advantage of the rapid adsorption of single stranded oligonucleotides to the Au-np. This adsorption has been documented using fluorescence quenching and Raman experiments {see Examples infra). These results are to some degree surprising because oligonucleotides are themselves commonly regarded as negatively charged species presenting negatively charged phosphate backbones that would be repelled by citrate. The rapid ss-DNA adsorption can be rationalized with a model where single stranded oligonucleotides can configure themselves with hydrophobic bases facing the Au-np. In this geometry, dipolar attraction can reduce the barrier to adsorption of ss-DNA and ss-RNA {see Examples infra). Double stranded oligonucleotides are unable to achieve an uncoiled geometry with exposed bases and, therefore, experience much larger repulsion by the ions on the Au-np surface. Consequently, they take much longer times to adsorb or do not adsorb to the Au-np at all under some conditions.
[0120] Once adsorbed, the single stranded oligonucleotides add negative charge density to the Au-np surface and act to enhance the stability of the colloid. It is therefore possible to protect the colloid from aggregation upon exposure to amounts of salt that would ordinarily screen the electrostatic repulsion between Au-np and induce aggregation. Hence, the gold will remain pink upon exposure to salt following exposure to ss-DNA or ss-RNA, while it will turn grayish-blue following exposure to ds-DNA or ds-RNA. This observation forms the basis for the colorimetric hybridization assay. The preferential adsorption of short ss-DNA probe sequences on Au-np can also be exploited to perform the fluorescent assay. When the ss-DNA probe is fluorescently tagged, adsorption to the metallic surface results in fluorescence quenching (Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer ^
Academic/Plenum Publishers, New York, NY (1999), which is hereby incorporated by reference in its entirety). However, if the probe binds to a target in the analyte solution, it is resistant to adsorption and its fluorescence persists indicating a match. The fact that these assays rely on the difference in electrostatic properties of ss-DNA and ds-RNA (or ds-DNA) distinguishes them from detection approaches using Au-nps covalently fiinctionalized with oligomers where hybridization is used to link the Au- nps (Elghanian et al., Science 277:1078-1081 (1997); Sato et al., J. Am. Chem. Soc. 125: 8102-8104 (2003), each of which is hereby incorporated by reference in its entirety). In the present work, the trial hybridization is performed separately from the assay and facilitates rapid duplex formation.
Materials and Methods for Examples 2-6
[0121] Gold particles with 13 run diameter were synthesized by reduction of
HAuCl4 (Gradar et al., Anal. Chem. 67:735-743 (1995), which is hereby incorporated by reference in its entirety). Briefly, 500 mL of 1 mM HAuCl4 was brought to a rolling boil with vigorous stirring. 50 mL of 38.8 mM sodium citrate was quickly added to the solution, and boiling was continued for 10 min. The heating mantle was then removed and the stirring was continued for an additional 15 minutes.
[0122] AU oligonucleotides were purchased from MWG Biotech, Inc. (High Point, NC) without further purification. Probes hybridize with targets in 10 mM phosphate buffer solution with 0.3 M NaCl for more than 5 minutes at room temperature or proper temperature.
[0123] The probes and targets employed in Example 2 are as follows:
Rhodamine red-labeled probe: AGGAATTCCATAGCT (SEQ ID NO: 25); and Target nucleic acid: AGCTATGGAATTCCT (SEQ ID NO: 26).
[0124] The probes and targets employed in Examples 3 and 4 are as follows:
Rhodamine red-labeled probe: AGGAATTCCATAGCT (SEQ ID NO: 25);
Complementary Target A: ACT AGGC ACTGTACGCCAGCTATGGAATTCCTT
AGCTATGAGATCCTTCG (SEQ ID NO: 9); Complementary Target B: GTTAGCTATGAGATCCTTCGTAGGCACTGTACGC
CAGCTATGGAATTCCT(SEQ ID NO: 10); and
Noncomplementary Target C: TGTGTTGAACCTGGTGAAGTTGTAATCTGGAA
CTTGTTGAGCAGAGGTTC (SEQ ID NO: 11). [0125] The probes and targets employed in Example 5 are as follows:
Rhodamine red-labeled probe: AGGAATTCCATAGCT (SEQ ID NO: 25); Complementary Target A': ACTAGGC ACTGTACGCCAGCTATCGAATTCCT TAGCTATGAGATCCTTCG (SEQ ID NO: 27); and Complementary Target B' : GTTAGCTATGAGATCCTTCGTAGGCACTGTAC GCO4 GCTATCGAATTCCT (SEQ ID NO: 28).
[0126] The probes and targets employed in Example 6 are as follows:
Rhodamine red-labeled probe 1 : CTGAATCCAGGAGCA (SEQ ID NO: 29); Complementary Target 1: the complement of probe 1; Cy5-labeled probe 2: TAGCTATGGAATTCCTCGTAGGCA (SEQ ID NO: 6); Complementary Target 2: the complement of probe 2; and Non-complement target: ATGGCAACTATACGCGCTAC (SEQ ID NO: 30). [0127] A fraction of hybridized solution was added to 500 μL of 17 nM gold colloid solution, and an additional 500 μL of the 0.1 M saline 10 mM phosphate buffer solution was added if without specific illustration. The fluorescence of this mixture was recorded immediately using either a fluorimeter, or a fluorescence microscope and camera. Fluorescence spectra were measured on a fluorimeter with excitation at 570 nm, and emission range from 585 to 680 nm, with slits set for 4 run bandpass unless specific illustration was given. Fluorescence images were recorded with a fluorescence confocol microscope equipped with notch filter and narrow bandpass interference filter. Fluorescence was excited by a 532 nm laser source.
Example 2 - Differential Fluorescence Quenching of Dye-Tagged Single- Stranded DNA and Double-Stranded DNA [0128] DNA oligonucleotides labeled with rhodamine red fluorescent dye covalently attached at the 5' end were used as probes. Several microliters of μM solutions of probe were exposed to the target sequences for trial hybridization in 10 mM phosphate buffer with 0.3 M NaCl. The hybridization solutions were added to colloidal gold suspensions and additional phosphate buffer saline solution was added to assist in stabilizing ds-DNA.
[0129] Figure 7A illustrates the result of a measurement comparing the photoluminescence from trial solutions with complementary and non-complementary targets. Fluorescence contrast larger than 100:1 was observed because unhybridized probes efficiently adsorb on the gold nanoparticles so that their fluorescence is quenched. The adsorption mechanism is entirely electrostatic, as discussed in Example 1 above. The adsorption and concomitant fluorescence quenching are irreversible. [0130] Addition of the trial hybridization solutions and salt to the gold colloid eventually cause aggregation of the colloid. The latter leads to precipitation so that the nanoparticles are no longer an effective quencher of the probe fluorescence. It is possible to protect the colloid against aggregation under conditions with sufficient salt to satisfy the duplex by using unrelated ss-DNA strands to stabilize the colloid. However, the data of Figure 7A illustrates that this is not necessary as long as the fluorescence measurements are made within about 15 minutes. [0131] Since relatively large volumes of solution are required for a typical fluorimeter, it is not practical to assess the sensitivity of the method using the same measurement protocol. Figure 7B illustrates measuring the fluorescence of a very small aliquot of the solution containing only 0.1 femtomoles of target and this is easily detected with a fluorescence microscope and camera. Since the method is essentially a null method, it stands to reason that it can be used in a relatively straightforward fluorescence detection down to fewer than 10 copies of target oligonucleotide (0.1 attomole) (Cao et al., Science 297:1536-1540 (2002), which is hereby incorporated by reference in its entirety).
Example 3 - Application to Long Target Sequences
[0132] For genomic analysis, it is desirable to detect specific sequences on much longer DNA targets than synthesized oligonucleotides. These could be derived directly from clinical samples or from samples that have been amplified using PCR. Figure 8 A is a proof of principle for detecting matches to parts of long targets. In spite of the fact that large portions of the target remain single stranded and will presumably have the electrostatic properties of ss-DNA, the assay can be used to determine whether these long targets contain sequences complementary to short dye- tagged probes. The reason adsorption and quenching are not observed in this case is that long ss-DNA sequences adsorb on the gold nanoparticles at a much slower rate, as noted in Example 7 herein. Thus, the technique is most practical when short dye- tagged probes (<25 mers) are used. Example 4 - Application to Mixtures of Target Sequences
[0133] Because the only requirement of the assay is that ss-DNA probes that do not hybridize to a target sequence in the analyte adsorb on gold and are quenched, the only constraint is that the amount of colloidal gold should be adequate to adsorb all of the probe DNA. Therefore, the assay can work to determine whether target strands are present even in complex mixtures of DNA oligonucleotides as demonstrated by the data of Figure 8B. In that case, 1% complementary target was mixed with 99% non-complementary target to verify the presence of the target sequence. The tolerance of the assay to mixtures, along with its sensitivity, provides the potential for it to be used without target amplification by PCR.
Example 5 - Single Base Mismatch Detection
[0134] It is simple to adapt the technique to detect single base mismatches by introducing a perfectly matched control and comparing the two with a stringency test. For illustrative purposes, two different target sequences that differ by a single base were used. One of these is perfectly matched to the dye-tagged probe. The only procedural difference is that, before introducing the two trial hybridization solutions to gold colloid, they are each held for 5 minutes at 46°C, a temperature above the melting temperature for the mismatch and below that for the perfect match. The mismatched strand dehybridizes, thereby releasing single stranded probe whose fluorescence can be quenched. The sample with a mismatch therefore exhibits much less photoluminescence than the perfectly matched target. Figure 9 shows the detection by one long target complementary to the probe in the middle portion and another long target complementary to the probe at one end. This procedure should be applicable to rapid detection of single nucleotide polymorphisms in genomic DNA, an exciting prospect for eliminating time-consuming and expensive gel sequencing procedures that are currently the standard protocol (Rolfs et al., PCR: Clinical Diagnostics and Research, Springer- Verlag, Berlin Heidelberg (1992), which is hereby incorporated by reference in their entirety). In practice, of course, one would use two different probe strand sequences and compare probes complementary to the wild type sequence to ones with single base mismatches at the targeted locations. Example 6 - Simultaneous Multiple Target Detection
[0135] The differential quenching assay can also be multiplexed to simultaneously look for several sequences on a single target or for several targets. Figure 10 illustrates this where two different probes with two different dyes are hybridized with a mixture of targets. If spectroscopic detection is used, it is plausible to imagine expanding this approach to 5 or 6 targets with conventional dyes and even more with semiconductor nanoparticle fluorophores that have spectrally sharp emission. This, of course, presumes that these do not perturb the essential electrostatics that is the basis of the method.
[0136] In summary, these experiments demonstrate a simple assay for DNA sequence recognition based on the difference in electrostatic properties of ss-DNA and ds-DNA. For certain salt concentrations, ss-DNA adsorbs on citrate-coated gold nanoparticles while ds-DNA does not and this fact can be exploited to differentially quench fluorescence of a dye-tagged ss-DNA probe. The method requires no target modification, uses only commercially available materials, works for analytes with mixtures of oligonucleotides, and can be applied to detection of single base mismatches. Perhaps the most attractive feature of the approach is its speed. The entire assay can be completed in less than 10 minutes because the hybridization step occurs in solution under optimized conditions and is separated from the detection step. A sensitivity to less than 0.1 femtomole of DNA oligonucleotides has been demonstrated, but, because the method is nearly a null method and relies on fluorescence detection, it is probably possible to improve this by several orders of magnitude. It is believed that the method has enormous promise for applications to pathogen detection, clinical analysis of SNPs, and biomolecular research.
Materials and Methods for Examples 7-9
[0137] All synthesized oligonucleotides were purchased from MWG Biotech,
Inc. (High Point, NC), and used without further purification. [0138] Colloidal solutions of gold nanoparticles were synthesized according to the procedure described in Grabar et al., Anal. Chem. 67:735-743 (1995), which is hereby incorporated by reference in their entirety). Briefly, 250 mL of 1 mM HAuCl4 (Alfa Aesar, Ward Hill, MA) was heated to its boiling point while stirring. 25 mL of 38.8 mM sodium citrate (Alfa Aesar, Ward Hill, MA) was added quickly to the boiling solution, while continuing to boil and stir the solution for another 15 minutes. The solution was cooled to room temperature and can be stored indefinitely for use. [0139] All photographs in this work were recorded with a Canon PowerShot S30 digital camera. Absorption spectra were recorded on a Perkin Elmer
UV/VIS/NIR spectrometer Lambda 19. Quartz cells with 2 mm or 5 mm path length were used and water was used as reference. Fluorescence spectra and intensities versus time were recorded on a Jobin-Yvon Fluorolog-3 spectrometer with excitation at 570 nm and emission at 590 nm, each with slits set for 4 nm bandpass. Quartz cells with 1 cm path length and front face collection were used for the fluorescence measurements.
Example 7 - Effects of Oligonucleotide Probe Length and Temperature on Adsorption of ss-DNA to Gold Nanoparticles [0140] To study effects of ss-DNA on gold nanoparticle aggregation, 300 μL gold colloid was mixed with 300 picomole 24 mer ss-DNA (5'-TGC CTA CGA GGA ATT CCA TAG CTA-3' (SEQ ID NO: 4)) in 10 μL of 10 mM PBS containing 0.2 M NaCl, and then 100 μL of 10 mM PBS containing 0.2 M NaCl was added. For comparison, 100 μL deionized water was mixed with 100 μL of 10 mM PBS containing 0.2 M NaCl with 300 μL gold colloid, respectively. Absorption spectra were recorded with 2 mm pathlength cells and photographs of the mixtures were taken. The spectra are stable with time.
[0141] To investigate sequence length dependent adsorption of ss-DNA to gold nanoparticles, 2 μL (2 μM) ss-DNAs with rhodamine red tags at the 5' end were added to 1000 μL of 13 nm gold colloid. The ss-DNA sequences were 10 mer (5'- CAG GAA TTC C-3' (SEQ ID NO: 5)), 24 mer (5'-TAG CTA TGG AAT TCC TCG TAG GCA-3' (SEQ ID NO: 6)), and 50 mer (5'-GAA CCT CTG CTC AAC AAG TTC CAG ATT ACA ACT TCA CCA GGT TCA ACA CA-3' (SEQ ID NO: I)). The fluorescence intensity versus time was recorded on the fluorimeter. [0142] To study the temperature dependence of ss-DNA adsorption, mixtures of 2 μL (100 μM) 50 mer ss-DNA and 300 μL of 13 nm gold colloid were heated to 220C, 450C, 7O0C, and 95°C for two minutes, respectively. Solutions of 300 μL of 10 mM PBS at 220C containing 0.2 M NaCl were added immediately and absorption spectra were measured with 5 mm pathlength cells.
[0143] To study the adsorption of short and long ss-DNA mixtures, 4 μL of 2 μM rhodamine red labeled 15 mer (5'-AGG AAT TCC ATA GCT-3' (SEQ ID NO: 8)) was mixed with each of three different 50 mers (sequences infra) in 10 mM PBS containing 0.3 M NaCl (4 μL at 2 μM concentration) for trial hybridization.
5'-AC TAG GCA CTG TAC GCC AGC TAT GGA ATT CCTTAG CTA TGA GAT CCT TCG-3' (SEQ ID NO: 9) complementary to the 15 mer at middle;
5'-GT TAG CTA TGA GAT CCT TCG TAG GCA CTG TAC GCC AGC TAT GGA ATT CCT-Υ (SEQ ID NO: 10) complementary to the 15 mer at end.
5'-TGT GTT GA ACCT GGT GAA GTT GTA ATC TGG AAC TTG TTG AGC AGA GGT TC-3' (SEQ ID NO: 11) non-complementary to the 15 mer. After 5 minutes for hybridization, each solution was mixed with 1 mL of 13 nm gold colloid and 0.4 mL additional 10 mM PBS containing 0.1 M NaCl and the resulting fluorescence spectrum was recorded on fluorimeter. Color photographs of the mixtures of 300 μL gold colloid, 6 μL (20 μM) hybridized DNA solution and 300 μL of 10 mM PBS containing 0.2 M NaCl taken with a Canon S-30 camera without unlabeled 15 mer of the same sequence. [0144] The color of gold colloid is very sensitive to the degree of aggregation of nanoparticles in suspension (Quinten et al., Surf. ScL 172:557 (1986); Lazarides et al., J. Phys. Chem. B 104:460 (2000); Storhoff et al., J. Am. Chem. Soc. 122:4640- 4650 (2000), which are hereby incorporated by reference in their entirety), and the aggregation can be easily induced with electrolytes such as salt (Hunter, Foundations of Colloid Science, Oxford University Press Inc., New York (2001); Shaw, Colloid and Surface Chemistry, Butterworth-Heinemann Ltd., Oxford (1991), which are hereby incorporated by reference in their entirety). This phenomenon can be easily monitored by absorption spectroscopy or by visual observation. Gold nanoparticles (13 nm in diameter) in aqueous solution are stabilized against aggregation by a negatively charged coating of citrate ions (Bloomfϊeld et al., Nucleic Acids: Structures, Properties, and Functions, University Science Books, Sausalito,
California (1999), which is hereby incorporated by reference in its entirety). As individual particles, gold nanoparticles have surface plasma resonance absorption peak at 520 nm (Figure 1 IA: red) and appear pink (Figure 1 IA, inset: left vial). Immediate aggregation of the gold nanoparticles occurs when enough salt is added to screen the electrostatic repulsion between the ion-coated gold nanoparticles. The result is a broad featureless absorption spectrum (Figure 1 IA: blue) and blue-gray color (Figure 1 IA, inset: middle vial) characteristic of the surface plasma resonance of gold nanoparticle aggregates (Quinten et al., Surf. Sd. 172:557 (1986); Lazarides et al., J. Phys. Chem. B 104:460-467 (2000); Storhoff et al., J. Am. Chem. Soc. 122:4640-4650 (2000), which are hereby incorporated by reference in their entirety). [0145] It was found that the salt no longer causes aggregation of the gold nanoparticles if enough ss-DNA is added to the gold colloid before addition of the salt that would otherwise cause aggregation. Under these circumstances, the gold colloid retains its absorption spectrum and color (Figure 1 IA: green and inset: right vial). The reason for the stabilization of the colloid is that the oligonucleotides adsorb and add negative charges to the gold nanoparticles that enhances their repulsion. This assertion is confirmed by fluorescence quenching experiments using rhodamine red- tagged ss-DNA (Figure HB). When the oligonucleotide adsorbs to the gold nanoparticle, the attendant proximity of the dye to the gold leads to fluorescence quenching (Maxwell et al., J. Am. Chem. Soc. 124:9606-9612 (2002); Dubertret et al., Nat. Biotech. 19:365-370 (2001), which are hereby incorporated by reference in their entirety). The fluorescence quenching experiments also show that the adsorption rate depends on sequence length, with shorter sequences sticking much more rapidly to the gold nanoparticle (Figure HB). hi addition, it is found that increasing temperature also results in faster adsorption (Figure 11C). Both the ss-DNA adsorption on gold nanoparticle and the gold nanoparticle aggregation inferred from the data in Figures 1 IA-D are irreversible.
[0146] The adsorption of ss-DNA on negatively charged gold nanoparticles is contrary to the conventional wisdom (Maxwell et al., J. Am. Chem. Soc. 124:9606- 9612 (2002); Graham et al., Angew. Chem. Int. Ed. 39:1061 (2000), which are hereby incorporated by reference in their entirety) since, in its native configuration, ss-DNA is coiled so that the hydrophilic negatively charged phosphate backbone is most exposed to the aqueous solution (Bloomfield et al., Nuclei Acids: Structures, Properties, and Functions, University Science Books, Sausalito, California (1999), which is hereby incorporated by reference in its entirety). The fact that ss-DNA sticks to gold nanoparticles, as well as the dependence on sequence length and temperature, can be explained with a simple picture derived from the theory of colloid science (Hunter, Foundations of Colloid Science, Oxford University Press Inc., New York (2001); Shaw, Colloid and Surface Chemistry, Butterworth-Heinemann Ltd., Oxford (1991), which are hereby incorporated by reference in their entirety). Both the gold nanoparticle and the ss-DNA attract counterions from the solution and are well described by electrical double layers as depicted schematically in Figure 12. In every case, there are attractive Van der Waals forces between the oligonucleotide and the nanoparticle. The electrostatic forces are due to dipolar interactions and depend on the configuration and orientation of the ss-DNA. When transient structural fluctuations permit short segments of the ss-DNA to uncoil and the bases face the gold nanoparticle, attractive electrostatic forces cause ss-DNA to adsorb irreversibly to the gold. The requisite fluctuations are more prevalent in short sequences since there is less of the chain remaining to enforce the coiled morphology. Hence, short ss-DNA oligonucleotides adsorb more quickly. Similarly, increases in temperature facilitate fluctuations that expose the bases and unwind the coiled structure to make the adsorption faster. Increases in temperature will also serve to break secondary structure in longer DNA chains thereby making the geometry of Figure 12 more easily accessible. [0147] The length dependent adsorption can be exploited to develop an assay appropriate to detection of PCR amplified DNA sequences that are typically several hundred base pairs in extent (Reed et al., Practical Skills in Biomolecular Sciences, Addison Wesley Longman Limited, Edinburgh Gate, Harlow, England (1998); Walker et al., Molecular Biology and Biotechnology, The Royal Society of Chemistry, Thomas Graham House, Cambridge, UK (2000), which are hereby incorporated by reference in their entirety). Short oligonucleotide "probes" can be designed with the idea that, when these are hybridized to the long strands, they will not adsorb rapidly on gold nanoparticle. They will therefore be unable to prevent salt- induced aggregation and the attendant color changes when there is a sequence match between the probe and part of the long strand. Alternatively, if the short probes are fluorescently labeled, their fluorescence will be quenched by adsorption on the gold nanoparticle unless they are "tied up" by hybridization to the long target strand. Figure 1 ID illustrates the proof of principle for each of these assays with synthesized 50 base oligonucleotide targets and rhodamine-labeled 15 base probe.
Example 8 - Detection of PCR-Amplified Target cDNA [0148] Genomic DNA obtained from Dr. Ming Qi of the University of
Rochester Medical Center was used as PCR template. Primers were synthesized oligonucleotides 5'-CCT GGG CAT TAA GGT TCC-3' (SEQ ID NO: 12) (forward) and 5'-TGG GAT TCT TCG GCT TCT TC-3' (SEQ ID NO: 13) (reverse). The specific region of KCNEl gene indicative of long QT syndrome was amplified in Promega PCR master mix (Promega, Madison, WI) with Tag DNA polymerase for 5 min at 95°C; 35 cycles of 30s at 95°C, 30s at 560C and 30s at 72°C; 10 min at 72°C and then held at 40C, yielding 189 bp PCR product.
[0149] Following these model experiments, simple colorimetric assays have been designed that address the critical issues that arise in the analysis of PCR amplified DNA. First, one can ascertain whether the amplified DNA contains the desired sequence by evaluating hybridization with the probes. Second, it is straightforward to identify SNPs in the amplified sequences. All of the experiments are performed on PCR product obtained from a clinical diagnosis laboratory without further purification. The sequence probed derives from genomic DNA taken in patient studies of a fatal cardiac arrhythmia called long QT syndrome (Priori et al., J. Interv. Card. Electr. 9:93 (2003), which is hereby incorporated by reference in its entirety). This condition has been associated with a mutation in KCNEl gene (Splawski et al., Circulation 102: 1178-1185 (2000), which is hereby incorporated by reference in its entirety). [0150] Current assays for point mutations on PCR amplified sequences involve time-consuming procedures, expensive instrumentation or both (Reed et al., Practical Skills in Biomolecular Sciences, Addison Wesley Longman Limited, Edinburgh Gate, Harlow, England (1998); Walker et al., Molecular Biology and Biotechnology, The Royal Society of Chemistry, Thomas Graham House, Cambridge, UK (2000); Rolfs et al., PCR: Clinical Diagnostics and Research, Springer- Verlag, Berlin Heidelberg (1992), which are hereby incorporated by reference in their entirety). The method takes less than ten minutes to verify amplification of the appropriate sequence and test for SNPs with the same thermal cycler used to do the PCR. To confirm amplification of the desired sequence, the protocol illustrated schematically in Figure 13A was followed. Two ss-DNA probes were chosen with sequences complementary to the desired PCR product that have melting temperatures lower than the primers and add these to the PCR product solution. The PCR amplified ds-DNA is dehybridized at 95°C to produce ss-DNA. These mixtures are annealed below the probe melting temperature so that the probes can hybridize with the PCR amplified sequence if it is present. At the same time, the unconsumed primers also bind to the PCR product since they have melting temperatures higher than those of the probes. As in the PCR process itself, competition for binding locations from rehybridization of the PCR amplified complement is negligible since it is slower for steric reasons. When gold colloid is exposed to this mixture, the salt in the hybridization solution causes immediate gold nanoparticle aggregation and a color change if the probes have hybridized to the amplified DNA target (Figure 13B, left vial). When the PCR product is not complementary to the probes or the PCR amplification fails altogether, the probes adsorb to the gold nanoparticles and prevent aggregation (Figure 13B, right vial).
Example 9 - Sequence Detection and Single Base-Pair Mismatch Detection of PCR-Amplifϊed Target cDNA [0151] For sequence detection, 8 μL of unmodified PCR product was mixed with 6 μL of 1 μM probe solution containing either two complementary probes or two non-complementary probes in 10 mM PBS containing 0.3 M NaCl. After 5 minute denaturation at 950C and 1 minute annealing at 5O0C, 60 μL gold colloid was added and photographs were taken. The probe sequences are as follows: 5'-CCT GTC TAA CAC CAC AG-3 ' (SEQ ID NO: 14) and 5'-CCA CAG CTT GGT CAG AA-3' (SEQ ID NO: 15) (complementary probes);
5'-ACC ACA CAC TGT CTC TC-3' (SEQ ID NO: 16) and 5'-CTG AGC ACA CTC AGT AC-3' (SEQ ID NO: 2) (non-complementary probes).
[0152] For single base-pair mismatch (SNP) detection, 8 μL PCR product was mixed with 6 μL of 1 μM probes overlapping the single-base mismatch, and 8 μL PCR product with 6 μL of 1 μM probes not overlapping the single base pair mismatch, respectively. The mixtures were heated at 950C for 5 minutes and annealed at 5O0C, 540C, and 580C for 1 minute, respectively, then 60 μL of gold colloid was added and a photograph was taken. The probes were as follows:
5'-CGG GAG ATG CAG GAG-3' (SEQ ID NO: 17) and 5'-ACG GCA AGC TGG AGG-3' (SEQ ID NO: 18) (no overlapped with SNP); 5'-CTT GCC GTC ACC GCT-3' (SEQ ID NO: 19) and 5'-CAG CGG TGA CGG CAA-3' (SEQ ID NO: 20) (overlapped with SNP).
[0153] Single base-pair mismatch detection requires a slightly different protocol since a single base mismatch still permits hybridization of the probe to the target sequence. The same concept as for specific sequence detection with the strategy depicted in Figure 14A was used. Four probes were selected that have the same melting temperature, lower than that of the PCR primers. The sequences were chosen to be complementary to the wild type sequence of the target. Two of the probes bound overlapping the position of the possible point mutation while two were used as controls and bound at locations that do not overlap the SNP under study. If a mutation exists on the target sequence, the probes covering the mutation will dehybridize at lower temperature than the control probes situated elsewhere in the sequence that are designed to be perfectly matched. At a temperature below the melting point of either sequence, the probes remain attached to the PCR amplified DNA and cannot prevent salt-induced gold nanoparticle aggregation (Figure 14B: a, b). Above the melting temperature of both perfect and mismatched sequences, dehybridization occurs for either and gold nanoparticle aggregation is prevented (Figure 14B: e, f). At temperatures above where the mismatched sequence dehybridizes but below where the perfectly matched sequence dehybridizes, color differences indicating the presence of a SNP are detected (Figure 14B: c, d). [0154] It has been demonstrated by these experiments that ss-DNA adsorbs to gold nanoparticle with a rate that is length and temperature dependent. In addition, adsorption of ss-DNA on gold nanoparticle can effectively stabilize the colloid against salt-induced aggregation. These observations were utilized to design a simple, fast colorimetric assay for PCR amplified DNA based strictly on the electrostatic properties of DNA. The approach obviates the need for gel electrophoresis and other complex sequencing procedures. It can be implemented with inexpensive commercially available materials in less than 10 minutes and no instrumentation beyond the programmable thermal cycler used for PCR is required. An important feature of the method is that, unlike chip-based assays (Fodor et al., Nature 364:555- 556 (1993); Chee et al., Science 274:610-614 (1996), which are hereby incorporated by reference in their entirety) or other approaches that utilize functionalized nanoparticles (Elghanian et al., Science 277:1078-1081 (1997); Taton et al., Science 289:1757-1760 (2000); Park et al., Science 295:1503-1506 (2002); Cao et al., Science 297:1536-1540 (2002); Maxwell et al., J. Am. Chem. Soc. 124:9606-9612 (2002); Dubertret et al., Nat Biotech 19:365-370 (2001); Sato et al., J. Am. Chem. Soc. 125:8102-8103 (2003), which are hereby incorporated by reference in their entirety), hybridization occurs under optimized conditions that can be regulated independent of the assay. The assay has also been applied to clinical samples of genomic DNA that screen for SNPs associated with a hereditary cardiac arrhythmia known as long QT syndrome. It is believed that this approach can replace some traditional analytical methods for post-processing of PCR amplified DNA and that it will find broad application.
Example 10 - RNA Detection Using Modified RNA Probe
[0155] For sequence detection, 2.4 μL of 100 μM 2'-o-methyl RNA probe was mixed with 2,4 μL of 100 μM RNA target containing either one complementary probe or one non-complementary probe in 10 mM PBS and 0.3M NaCl solution.
After 2 minute denaturation at 95°C and 30 minute annealing at a temperature below the melting temperature of probe, 200 μL gold colloid was added and photographs were taken. The amount of RNA and gold colloid can be increased or decreased accordingly. In this case, a relatively large amount RNA and gold was used to measure visible spectra on regular spectrometer.
[0156] The probe and target sequences are as follows:
2'-o-methyl RNA probe: AGGAAUUCCAUAGCU (SEQ ID NO: 21); perfect matched target: AGCUAUGGAAUUCCU (SEQ ID NO: 22); and non-complementary target: CGAUCACGAGAUCGA (SEQ ID NO: 23).
[0157] For single base-pair mismatch (SNP) detection, 2.4 μL of 100 μM T- o-methyl RNA probe 1 (perfectly matching with target) was mixed with 2.4 μL of 100 μM targets and 2.4 μL of 100 μM 2'-o-methyl RNA probe 2 ( single mismatch with target) with 2.4 μL of 100 μM target respectively. The mixtures were heated at 950C for 2 minutes and annealed at 500C and 6O0C for 30 minutes, respectively, then 200 μL of gold colloid was added and a photograph was taken. [0158] The probe and target sequences are as follows:
2'-o-methyl RNA probe: AGGAAUUCCAUAGCU (SEQ ID NO: 21); perfect matched target: AGCUAUGGAAUUCCU (SEQ ID NO: 22); and single mismatched target: AGCUAUAGAAUUCCU (SEQ ID NO: 24). [0159] As shown in Figures 15A-B, an RNA probe can be used to effectively discriminate between a SNP and a wild-type sequence.
Example 11 - Immuno-PCR Protocol
[0160] A detection protocol employing a capture antibody and a biotinylated detection antibody coupled via streptavidin to a biotinylated DNA molecule can be employed in detecting the presence of an antigen using standard immuno-PCR procedures. If the antigen is present, PCR will result in amplification of the biotinylated DNA molecule. Assuming the antigen was present, the amplified PCR product will be detected by colorimetric or fluorimetric detection methods described in the above examples.
Example 12 — Detection of Target Nucleic Acid Using Immobilized Citrate- Coated Gold Nanoparticles
[0161] As illustrated in Figure 16, citrate-coated gold nanoparticles (prepared as described above) were attached to the surface of glass beads, the beads were loaded into a column, and then the hybridization product was introduced into the column to collect the eluted solution (which contains the double stranded DNA labeled with fluorophores). This approach effectively solved the contrast problem identified above. This process can be repeated more than once to optimize the results. [0162] The detailed procedure included the following steps:
1. Cleaning glass beads: Glass beads of 1 mm diameter were washed with piranha solution for 20 min, rinsed with clean water thoroughly, and then dried on a hot plate. 2. Coating glass beads with amino-group terminal molecules: glass beads were immersed in aminopropyl triethoxysilane (APTES) in toluene solution for 30 min, and then washed thoroughly with toluene. The APTES-modifϊed glass beads were then baked in an oven at 1000C. 3. Coating glass beads with gold nanoparticles: APTES-modified glass beads were immersed in gold colloid for 30 min, then rinsed with clean water thoroughly, dried on a hot plate, and cooled to room temperature for use.
4. Detection: fiuorescently labeled DNA probe was allowed to hybridize with its complementary target or non-complementary target in hybridization buffer solution for more than 5 min. The hybridization solution was then passed through the column (loaded with the modified glass beads coated with gold nanoparticles). The eluent was then collected and examined for fluorescence measurement. In this experiment, the DNA sequences were as follows: probe: 5'-AGG AAT TCC ATA GCT-S' (SEQ ID NO: 8) c-target: 5'-AGC TAT GGA ATT CCT-3' (SEQ ID NO: 37) nc-target: 5'-TAA CAA TAA TCC CTC-3' (SEQ ID NO: 38)
[0163] 100 picomoles rhodamine red labeled probe hybridized with the same amount of its complementary target (c-target) or non-complementary target (nc- target) in 10 mM PBS containing 0.3 M NaCl for more than 5 min. Detection of ds- DNA and ss-DNA is illustrated in Figure 17. The red (upper) curve was recorded from the hybridization solution containing c-target after going through beads coated with gold nanoparticles, whereas the green (lower) curve was recorded from the hybridization solution containing nc-target after going through beads coated with gold nanoparticles.
Example 13 — Formation of Citrate- or Polyanion-Coated Glass Beads
[0164] Small anion glass beads can be made by exposure of glass beads to aqueous solution containing the anions. Under appropriate conditions of temperature and pH, the glass surface will be effectively coated with the anions. Polyanionic coatings of a wide variety of substrates can be accomplished by simply dipping substrates in polyelectrolyte solutions according to the methods of Shiratori and Rubner, "pH-dependent Thickness Behavior of Sequentially Adsorbed Layers of Weak Polyelectrolytes," Macromolecules 33(11):4213-4219 (2000), which is hereby incorporated by reference in its entirety. Example 14 - Formation of Patterned Charged Films on a Substrate
[0165] Patterned charged films to be used to concentrate ds-nucleic acid can be fabricated in accordance with the procedures described in Zhang et al., "Particle Assembly On Patterned "Plus/Minus" Polyelectrolyte Surfaces Via Polymer-On- Polymer Stamping," Langmuir 18(11):4505-4510 (2002), which is hereby incorporated by reference in its entirety.
Example 15 - Separating Citrate-Coated Gold Nanoparticles (and ss-DNA
Bound Thereto) From ds-DNA in Solution Via Crashout Method [0166] The crashout method involves first using the interactions in solution to adsorb the ss-DNA preferentially on the gold (or other negatively charged) nanoparticles, but then removing the nanoparticles and ss-DNA bound thereto, leaving the ds-DNA (target) to be analyzed. This is called the "crashout" method since it involves removing the nanoparticles from solution rather than removing the ss-DNA from solution.
[0167] The protocol for this method is similar to the fluorescence method described above. The analyte was first hybridized against the fluorescently tagged probe with sequence complementary to the target (whose presence is being screened). The hybridization solution was then introduced into gold colloid, and followed by the addition of salt solution. (While in the fluorescence method the purpose of the salt was simply to further stabilize ds-DNA, in the crashout method its primary purpose is to aggregate the gold nanoparticles so that they can be removed from, i.e., crashed out of, solution.) The salt concentration should be provided within the range of about 0.1- 1 M, because too much salt will permit the repulsion of the nanoparticle coating to be screened so that ds-DNA will adsorb, whereas too little salt will not cause the gold to aggregate.
[0168] Of the above procedures, the salt-induced aggregation and centrifugation is described in greater detail. To 500 μL of gold colloid (13 nm particles, 17 nM solution), 100 uL of ss-DNA (or ds-DNA) solution was added (0.2 M salt, 10 mM PBS). A red to blue color change characteristic of gold aggregation was observed. Following aggregation, the mixture was centrifuged for 2 minutes. The clarified solution was transferred via pipette into a polystyrene cuvette and then scanned in a fluorimeter. The results are shown in Figure 18, which demonstrates that a contrast greater than 10 was achieved. Because this is an early result, it is believed that significant improvements in contrast will be achieved as the protocol is optimized. One possibility for achieving this improvement it to apply the method more than once. [0169] It is also contemplated that anion- or polyanion-coated non-metallic nanoparticles can be substituted for the colloidal gold nanoparticles in the crash out method given that quenching of fluorescence is no longer relied upon for signal detection perse.
Example 16 - Other Tagging Approaches Used With Beads or Crashout Separation Techniques
[0170] Not only do the immobilized beads and crashout methods solve the contrast problem, but they also allow for the use of other labels besides fluorescent tags. Two suitable labels are radioactive tags and electrochemical ("redox") tags. Following separation of the nanoparticles that remain in solution from aggregates, electrochemical detection can be carried out using either cyclic voltammetry (De-los- Santos-Alvarez, Anal. Chem. 74:3342-3347 (2002), which is hereby incorporated by reference in its entirety), stripping potentiometry (Wang et al., Anal. Chem. 73:5576- 5581 (2001), which is hereby incorporated by reference in its entirety), square wave voltammetry (Mugweru et al., Anal. Chem. 74:4044-4049 (2002), which is hereby incorporated by reference in its entirety), differential voltammetry (Olivira-Brett et al., Langmuir 18:2326-2330 (2002), which is hereby incorporated by reference in its entirety), and AC impedance spectroscopy (Ruan et al., Anal. Chem. 74:4814-4820 (2002); Yan et al., Anal. Chem. 73:5272-5280 (2001); Patolsky et al., Langmuir 15:3703-3706 (1999); Patolsky et al., J. Am. Chem. Soc. 123:5194-5205 (2001), each of which is hereby incorporated by reference in its entirety). [0171] It is expected that detection of 10"17 or 10"18 moles of target can be achieved easily using the beads method and electrochemical tagging of probe nucleic acid by ferrocene. The electrical method is particularly interesting in that the instrumentation needed to sense presence of the redox tags can be miniaturized into small devices, for example those that exploit PCR-on-a-chip where electronic detection could be integrated onto the same chip. Example 17 - Concentration of Eluted ds-Nucleic Acid for Ultrasensitive Assays
[0172] For most applications, the part of the analyte that comes through the column can be easily analyzed for fluorescence (or radioactivity or electrochemical activity). However, to perform ultrasensitive detection of just a few copies of nucleic acid, it is possible to concentrate the analyte that elutes from a column (or other separation procedure). Concentrating the analyte can potentially reduce or eliminate the need for PCR amplification.
[0173] Once the column has separated out unbound probe, there is no reason not to collect all of the ds-nucleic acid. This can be achieved by using a positively charged spot on a negatively charged surface so that the ds-nucleic acid will stick in a predefined location for analysis. In the fluorescence case, the charged spot can be prepared using a polycation (e.g., polyamine), and detection equipment (e.g., fluorescence microscope) can be focused on the predefined location. In the electrochemical case, the charged spot can be a micro-electrode (e.g., gold, platinum, etc.) functionalized with a monolayer whose end group is positively charged, e.g., NH2.
[0174] A patterned polyelectrolyte can be made as follows. First, a negatively charged surface to which DNA will not adhere is formed on a glass slide by standard electrostatic self-assembly techniques. For example, a multilayer structure can be formed with a PAA (polyacrylic acid) top layer. A PDMS stamp can be fabricated with a predefined recessed region the size of the desired microscope focus. The stamp is inked with a monolayer to pattern that surface to be hydrophobic everywhere except for the place where the stamp is recessed. An ink made of ODA (CH3(CHa)17NH2) in an organic solvent is suitable for this purpose; the applicants have previously demonstrated use of this ink. The amine is attracted to the carboxylate terminations of the PAA surface, thereby transforming the hydrophilic PAA to hydrophobic in the region where the ink layer is applied. A positively charged electrolyte can be applied to the resulting patterned surface, and the electrolyte will only stick where there is no ODA. The ODA can then be removed by rinsing with organic solvent, leaving the patterned charged surface. Application of the processed analyte, where only tagged ds-nucleic acid should remain, will allow the DNA to be concentrated onto the positively charged spot for analysis. Example 18 - Formation of Positively Charged Microparticles
[0175] Polycationic polystyrene or silica microspheres can be made by exposure of microspheres to aqueous solutions containing cations. Under proper conditions of temperature and pH, the microsphere surface will effectively be coated with the cations. Polycationic coatings of a wide variety of substrates can be accomplished by simply dipping substrates in polyelectrolyte solutions.
Example 19 - Concentration of Eluted ds-Nucleic Acid for Ultrasensitive Assays
[0176] Polycationic microparticles can be introduced into eluent that comes through a separation column of the type described in Example 17 above. The microparticles will adsorb labeled ds-DNA onto a small volume. Upon collection, the microparticles can be introduced onto a negatively biased electrode for analysis using a confocal microscope.
Materials and Methods for Examples 20-21
[0177] Synthesis ofAu-nps: Hydrogen tetrachloroaurate (III) (HAuCl43H2O),
99.99% and sodium citrate (Na3C6H5O7IH2O), 99%, were purchased from Alfa Aesar and used without further purification. Gold colloid, an aqueous suspension of Au-nps stabilized against aggregation by sodium citrate, was prepared as described elsewhere (Grabar et al., "Preparation and Characterization of Au Colloid Monolayers," Anal. Chem. 67:735-743 (1995), which is hereby incorporated by reference in its entirety). Briefly, 250 mL of 1 mM HAuCl4 (Alfa Aesar, Ward Hill, MA) aqueous solution was heated to its boiling point while stirring. After adding 25 mL of 38.8 mM sodium citrate (Alfa Aesar, Ward Hill, MA) in water rapidly to the boiling solution, boiling and stirring continued for 15 minutes. The solution was then cooled to room temperature for use. The gold nanoparticle diameters were measured by TEM to be ~13 nm, which is consistent with their absorption spectrum (maximum at 520 nm). The concentration of the gold colloid was about 17 nM. [0178] Selection and preparation of oligonucleotide targets and probes: A 2- o-methyl RNA oligonucleotide (5'-AGG AAU UCC AUA GCU-3 ', SEQ ID NO: 34) was synthesized and purified by IDT (Coralville, IA) to be used as a probe sequence for the colorimetric assay. Three RNA sequences with the same length as the probe were used as targets. These were synthesized and purified (RNase-free HPLC purification, RNA oligos of greater than 85% foil length product) by IDT. One sequence (c-target) was complementary to probe, the second (mc-target: 5'-AGC UAU AGA AUU CCU-3 ', SEQ ID NO: 35) had a one base-pair mismatch with the probe and the third (nc-target: 5'-CGA UCA CGA GAU CGA-3', SEQ ID NO: 33) is not complementary to the probe. For the fluorescent assay, rhodamine red labeled DNA were used as probes (wild-type probe: rhodamine red-5'-AGG AAT TCC ATA GCT-3', SEQ ID NO: 8, and mutant probe: rhodamine red-5'-AGG AAT GCC ATA GCT-3', SEQ ID NO: 36). Rhodamine red labeled DNA sequences were purchased from MWG Biotech (High Point, NC). 2'-ACE protected 50 mer RNA50a and RNA50b were purchased from DHARMACOM RNA Technologies (Lafayette, CO). These two sequences only have a single base difference in their sequences (RNA50a (/RNA50b): 5'-ACU AGG CAC UGU ACG CCA GCU AUG GA(/C)A UUC CUU AGC UAU GAG AUC CUU CG-3', SEQ ID NOS: 31-32, respectively. RNA50a contains a sequence perfectly matched with the wild-type probe while the analogous segment of RNA50b has a single base-pair mismatch with the wild-type probe. Conversely, the target sequence on RNA50b is perfectly matched with the mutant probe so that the analogous segment of RNA50a has a single base-pair mismatch with the mutant probe. [0179] RNA and DNA solutions with concentrations of salt and phosphate buffer as specified in the text were made. The requisite potassium phosphate (monobasic, anhydrous 99.999%) and sodium phosphate (dibasic, anhydrous, 99.999%) were obtained from Aldrich Chemical (Milwaukee, WI) and used as supplied. Sodium chloride crystals were purchased from Mallinckrodt (Hazelwood, MO). [0180] Deprotection of 2 '-ACE protected RNA: Prior to attempted hybridization, 2'-ACE protected RNA was deprotected according to the procedure provided by the manufacturer and used without further purification. Deprotection involves centrifugation for 2 minutes, adding 400 μL of deprotection buffer to the tube of RNA and completely dissolving the resulting RNA pellet. This solution was spun for 10 seconds, centrifoged for 10 seconds and incubated at 60°C for 30 minutes. The sample was then dehydrated using a SpeedVac before use. [0181] Hybridization: A trial hybridization solution containing 20 picomoles of each probe and target sequences was made in 10 mM phosphate buffer solution (PBS) containing 0.3 M NaCl. To break any secondary structure in the RNA target and allow hybridization with the probe, the trial solution was heated to 95°C for 3 minutes and then cooled to an appropriate temperature for the desired assay for 1 minute. The temperature used for simple sequence detection was typically ambient while single base mismatch detection requires that hybridization takes place at a temperature between the melting temperature of the mismatch and that of the perfect match. In performing the assays below, the gold colloid was used at ambient temperature regardless of the temperature of the trial solution. [0182] Colorimetric Detection: 50 μL gold colloid solution was added to 10 μL trial hybridization solution and the color of the mixture is viewed immediately. Photographs were recorded with a Canon S-30 digital camera. [0183] Fluorescence Detection: 5 μL trial hybridization solution was added to 500 μL gold colloid, then mixed with 500 μL of 10 PBS containing 0.3 M NaCl. The fluorescence spectrum of the mixture was recorded within 2 minutes after mixing in a fluorimeter (Fluorolog 3, Jobin Yvon) with excitation at 570 ran over the range of emission wavelengths from 585 to 680 nm. Spectrometer slits were set for 4 nm bandpass. Traces of photoluminescence versus time were recorded with at 590 nm near the rhodamine emission maximum. The large solution volumes were used to facilitate measurements with a fluorimeter designed for centimeter pathlength cuvettes and fluorescence was efficiently collected from only ~ 1 % of the sample volume. The sensitivity of the fluorescent assay, as discussed in the preceding Examples, was therefore greatly underestimated.
Example 20 - Colorimetric Detection of RNA Oligonucleotides [0184] Figures 19A-D are images taken immediately after mixing trial hybridization solutions with gold colloid. The quantity of salt in the hybridization solution was adequate to cause Au-np aggregation in the absence of RNA. Each vial contains 10 μL trial hybridization solution that contains 10 mM PBS and 0.3 M NaCl and 50 μL gold colloid. In the hybridization solution, there were 20 picomoles of RNA probe and target. For the probe, 2'-o-methyl RNA was used because of its high stability (Majlessi et al., "Advantages of 2'-O-methyl Oligoribonucleotide Probes for Detecting RNA Targets," Nucleic Acids Res. 26:2224-2229 (1998), which is hereby incorporated by reference in its entirety). Complementary (c), single base mismatched (mc), and unrelated (nc) target sequences were used in the left, center and right vials, respectively. All trial hybridization solutions were heated at 950C for 3 minutes, then annealed for 1 minutes at the specified temperatures (A: 20°C, B: 50°C, C: 59°C and D: 64°C). In Figures 19A and 19B, the c and me targets presented a gray color while the nc target appeared light pink. This result indicates that c-target and mc-target hybridize with probe and form ds-RNA below 50°C. The ds-RNA does not absorb to Au-nps so that the salt in the hybridization solution causes Au-np aggregation and color change. Since the nc target is not complementary to the probe, both nc-target and probe remain single stranded. They therefore adsorb rapidly to the Au-nps and stabilize them against salt-induced aggregation so that the gold colloid remains pink. When the annealing temperature was elevated to 59°C (Figure 19C) prior to mixing with Au-np, the mixture containing c-target again turned gray but the mixture containing mc-target remained pink because 59°C is above the melting temperature (Tm) of the mc-target but lower than that of the c-target. At 64°C (Figure 19D), none of the targets can hybridize to the probe and all of the solutions appear pink. It is practical to heat only the trial hybridization solutions, but allow the gold colloid to remain at 20°C because hybridization under the conditions in the colloid is much slower than adsorption to the Au-np. At the same time, there is adequate salt in the mixture to maintain the stability of the double strand for longer than the Au-np takes to aggregate. The Au-np aggregation is irreversible.
[0185] Color changes, though detectable by human eye, can be more sensitively and quantitatively monitored by absorption spectra (Figure 20). These exhibit the characteristic isolated Au-np spectra in cases where aggregation does not occur and the broad red tail associated with aggregates when the salt is able to cause aggregation. Substantial changes in salt-induced aggregation behavior as for Figures 1 and 2 are observed for > 10 oligonucleotides (15 mers) per Au-np. Remarkably, this corresponds to occupying only ~ 1 % of the Au-np surface area with ss-RNA. Because of the enormous extinction coefficient (~ 107 lit-mol^cm"1) associated with the gold nanoparticles, the color of 17 nM Au-np solutions is easily detected by eye in 10 μL droplets or by using an absorption spectrometer with 100 μm pathlength sample cells. The data of Figures 19-20 were recorded with approximately 40 single strands (or 20 double strands) of RNA per Au-np, illustrating that subpicomole target RNA detection by visual inspection is possible.
Example 21 - Fluorescent Detection of RNA Long-mers [0186] There are some limitations on colorimetric detection that can be ameliorated by using the fluorescent assay described above. Since traditional absorption spectroscopy is, by its nature, not a null experiment, its sensitivity is limited. Moreover, a number of ambiguities arise in the context of using the colorimetric method illustrated in the preceding examples. For example, it is easy to imagine circumstances where the quantities of target and probe differ so that the trial hybridization solution contains both single and double strands, hi addition, situations where the length of the probe does not match that of the target leaves a single stranded overhang on a double stranded complex. Using the fluorescent assay, these practical difficulties do not arise. Since only the fate of the fluorescently tagged probe strand is monitored, unmatched targets do not affect the assay. When the probe sequence hybridizes with a sequence in the analyte, it will be protected from adsorption and the concomitant fluorescence quenching. Thus, as long as there is adequate concentration of Au-nps to adsorb all of the probe oligonucleotides, the presence of fluorescence indicates the presence of the target sequence in the analyte. A null result should exhibit no fluorescence. The null character of the measurement, high sensitivity of fluorescence detection, and ability to work in complex mixtures of target make this a powerful assay for DNA detection.
[0187] For RNA sequence detection, DNA sequences were labeled with rhodamine red as probes because RNA oligonucleotides are difficult to fluorescently label. Long synthetic targets (50 bases) with secondary structure were used to simulate genomic RNA, and 15 base probes were used to assay for a complementary sequence on the targets. The hybridization solution was heated to 940C for 3 minutes to break up secondary structure and annealed for 1 minute at a lower temperature. As demonstrated for the colorimetric method, single base mutations can be detected by careful choice of annealing temperature for hybridization. The duplex formed from a probe and mutant target has a lower melting temperature than the duplex formed from the probe and wild-type target. Hybridization at a temperature between the melting temperature of these two duplexes will only result in duplex formation for wild-type targets. When the probe hybridizes with the RNA target, it does not adsorb to Au-nps and its fluorescence persists. The result of this experiment is shown in Figure 21 where 15 base probes were used to detect wild-type 50-mers, while 50-mers with a single base difference overlapping the probe sequence do not yield appreciable signal. [0188] For practical purposes, it is desirable to detect target RNA sequences in a complex mixture of oligonucleotides. Because the fluorescent method is structured so that luminescence will be observed as long as the tagged probes hybridize with some component of the analyte, it is well suited to mixtures. To demonstrate this feature, short RNA sequences non-complementary to the probes were added to the trial hybridization solution at concentrations 10 times that of the target. Figure 22 depicts the time course of the luminescence after mixing the trial hybridization solution with Au-nps as monitored at the wavelength maximum of the fluorescence in an experiment analogous to that of Figure 21, but where the hybridization solution contains 5 picomoles probe, 5 picomoles target and 50 picomoles of short RNA noncomplementary segments. Each combination of wild type and mutant probe and targets are illustrated at an annealing temperature below the wild type melting temperature. These data verify that choice of the probe sequence perfectly matching the mutant target will, of course, result in much more fluorescence than the wild-type probe sequence when exposed to the mutant target. An important implication of Figure 22 is that the fluorescent assay can tolerate substantial amounts of RNA degradation into short sequences as often occurs. As long as there is an adequate concentration of Au-np, these do not interfere with adsorption of unhybridized probes and the attendant fluorescence quenching essential to the assay. [0189] The dynamics reflected in Figure 22 are important in the performance of the assay since the fluorescence should be evaluated at a time long enough to allow for adsorption of the unhybridized probes but short enough relative to the lifetime or adsorption rate of the hybridized complex formed between probe and target. Under the conditions of Figure 22, the adsorption of unhybridized probes on the Au-np is very rapid and occurs prior to the beginning of the trace. The subsequent slow decay seen in Figure 22 has several possible explanations. The complex formed between the probe and target may not be perfectly stable in the gold colloid and may slowly dehybridize. Even if it does not, single stranded portions of the long target strand may adsorb and bring the probe fluorophore close to the Au-np so that quenching is observed. Finally, there can be slow adsorption of even perfect duplexes onto the gold at the salt concentrations used in the experiment. It has been demonstrated empirically that the duplex sticks rapidly to Au-np at high salt concentrations where the electrostatic repulsion between the citrate coating on the Au-np and the phosphate backbone is heavily screened.
[0190] The above examples demonstrate a simple approach to detection of specific RNA sequences based on the differential adsorption rates for single and double stranded oligonucleotides onto Au-nps. A colorimetric assay for target RNA sequences with 2-o-methyl RNA probes and a fluorescent assay based on hybridization of target RNA with fluorescently labeled DNA probes have been developed. The assays require only commercially available reagents. A key strength of the methods is that the hybridization step is completed independent of the assay so that it can be performed under optimal conditions for rapid, efficient hybridization. Each assay therefore takes less than 10 minutes so that issues concerning RNA instability are minimized. Single base mismatches between probe and target sequences are easily detected with high contrast. The fluorescent assay is particularly promising since it is effective even for complex target mixtures and in cases where the probe and target have quite different length. This will allow its use in searching for target sequences in samples of genomic RNA. These methods will find wide application in molecular biology and clinical diagnosis.
[0191] Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

Claims

WHAT IS CLAIMED:
1. A method for detecting presence or absence of a target nucleic acid in a test solution comprising: combining at least one single-stranded oligonucleotide probe with a test solution potentially including a target nucleic acid to form a hybridization solution, wherein the at least one single-stranded oligonucleotide probe and the test solution are combined under conditions effective to allow formation of a hybridization complex between the at least one single-stranded oligonucleotide probe and any target nucleic acid present in the test solution; exposing the hybridization solution to a plurality of negatively charged nanoparticles under conditions effective to allow any single-stranded oligonucleotide probe or non-target nucleic acid that remains unhybridized after said combining to associate electrostatically with the plurality of negatively charged nanoparticles; separating the plurality of negatively charged nanoparticles from the hybridization solution after said exposing; and determining whether the at least one single-stranded oligonucleotide probe has hybridized to target nucleic acid.
2. The method according to claim 1, wherein the negatively charged nanoparticles comprise anion-coated nanoparticles.
3. The method according to claim 2, wherein the anion is selected from the group of citrate, acetate, carbonate, dihydrogen phosphate, oxalate, sulfate, and nitrate anions.
4. The method according to claim 2, wherein the nanoparticle is formed of a conductive metal.
5. The method according to claim 4, wherein the conductive metal is gold, silver, or platinum.
6. The method according to claim 2, wherein the nanoparticle is formed of a non-conductive material.
7. The method according to claim 6, wherein the non-conductive material is glass.
8. The method according to claim 6, wherein the non-conductive material is coated by a polyanion.
9. The method according to claim 8, wherein the polyanion is selected from the group of poly(2-acrylamido-2-methyl-l-propanesulfonic acid), poly(acrylic acid), poly(anetholesulfonic acid), poly(anilinesulfonic acid), poly(sodium 4-styrenesulfonate), poly(4-styrenesulfonic acid), and poly(vinylsulfonic acid).
10. The method according to claim 1 , wherein the plurality of negatively charged nanoparticles are immobilized on a surface.
11. The method according to claim 10, wherein said exposing comprises introducing the hybridization solution to the surface, and said separating comprises recovering the eluted hybridization solution from the surface.
12. The method according to claim 10, wherein the surface is a glass surface.
13. The method according to claim 12, wherein the glass surface comprises a plurality of glass beads.
14. The method according to claim 1 wherein said exposing comprises: adding to the hybridization solution a salt solution comprising a concentration of salt that is effective to cause aggregation of the negatively charged nanoparticles.
15. The method according to claim 14 wherein said separating comprises: centrifuging the hybridization solution under conditions effective to remove from the solution aggregates of the negatively charged nanoparticles.
16. The method according to claim 1 , wherein the plurality of negatively charged nanoparticles are magnetic.
17. The method according to claim 16, wherein said separating comprises: exposing the hybridization solution to a magnetic field that removes the magnetic, negatively charged nanoparticles from the hybridization solution.
18. The method according to claim 1, further comprising: concentrating double-stranded nucleic acid molecules onto a charged solid surface.
19. The method according to claim 18, wherein the charged solid surface comprises a negatively charged surface having a location on the surface that is positively charged.
20. The method according to claim 1, wherein the oligonucleotide probe comprises a label.
21. The method according to claim 20, wherein the label is a fluorophore, radiolabel, or redox electrochemical.
22. The method according to claim 21 , wherein the label is a fluorophore and said determining comprises detecting fluorescence of the fluorophore in the hybridization solution after said separating.
23. The method according to claim 21 , wherein the label is a radiolabel and said determining comprises detecting radioactivity of the radiolabel in the hybridization solution after said separating.
24. The method according to claim 21, wherein the label is a redox chemical and said determining comprises detecting electrochemical activity reflecting the presence of the redox chemical of the hybridization solution after said separating.
25. A method of detecting a pathogen in a sample comprising: obtaining a sample that may contain nucleic acid of a pathogen; and performing the method of claim 1, wherein said determining that the at least one single-stranded oligonucleotide probe has hybridized to the target nucleic acid indicates presence of the pathogen.
26. The method according to claim 25 wherein the nucleic acid isolated from the sample is RNA and the target nucleic acid is RNA.
27. The method according to claim 25, wherein the nucleic acid isolated from the sample is DNA and the target nucleic acid is DNA.
28. A kit comprising: a first container comprising a plurality of negatively charged nanoparticles; and a second container comprising a salt solution comprising a concentration of salt that is effective to cause aggregation of the negatively charged nanoparticles.
29. The kit according to claim 28, wherein the negatively charged nanoparticles comprise anion-coated nanoparticles.
30. The kit according to claim 29, wherein the anion is selected from the group of citrate, acetate, carbonate, dihydrogen phosphate, oxalate, sulfate, and nitrate anions .
31. The kit according to claim 29, wherein the nanoparticle is formed of a conductive metal.
32. The kit according to claim 31 , wherein the conductive metal is gold, silver, or platinum.
33. The kit according to claim 29, wherein the nanoparticle is formed of a non-conductive material.
34. The kit according to claim 33, wherein the non-conductive material is glass.
35. The kit according to claim 33, wherein the non-conductive material is coated by a polyanion.
36. The kit according to claim 35, wherein the polyanion is selected from the group of poly(2-acrylamido-2-methyl-l-propanesulfonic acid), poly(acrylic acid), poly(anetholesulfonic acid), poly(anilinesulfonic acid), poly(sodium 4-styrenesulfonate), poly(4-styrenesulfonic acid), and poly(vinylsulfonic acid).
37. The kit according to claim 28, wherein the plurality of negatively charged nanoparticles are immobilized on glass beads and the beads are retained within a column.
38. The kit according to claim 28 further comprising one or both of: a third container comprising at least one single-stranded oligonucleotide probe complementary to a target nucleic acid; and a fourth container comprising a hybridization solution.
39. The kit according to claim 38 further comprising one or more centrifugation tubes.
40. The kit according to claim 28, wherein the plurality of negatively charged nanoparticles are magnetic.
41. The kit according to claim 28 further comprising: a negatively charged solid surface comprising a location of the surface that is positively charged.
42. The kit according to claim 28, wherein the oligonucleotide probe comprises a label.
43. The kit according to claim 42, wherein the label is a fluorophore, radiolabel, or redox electrochemical.
44. The kit according to claim 28 further comprising a filter.
45. A kit comprising: a container comprising a plurality of negatively charged nanoparticles immobilized on glass beads; and instructions for performing an assay for separation of single-stranded nucleic acids from double-stranded nucleic acids, and detection of double-stranded nucleic acids passed over the plurality of negatively charged nanoparticles.
46. A detection device for performing the method according to claim 1.
47. A method of detecting a single nucleotide polymorphism (SNP) in a target nucleic acid molecule comprising: obtaining a sample comprising single-stranded nucleic molecules; and performing the method of claim 1 at temperatures above and below the melting temperature of target molecule comprising the SNP; wherein said determining comprises detecting whether the ds- hybridization complex is present after said separating when said combining is performed below but not above the melting temperature of the target molecule comprising the SNP.
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