Anal. Chem. 2004, 76, 5414-5417
DNA Sequence Detection Using Selective Fluorescence Quenching of Tagged Oligonucleotide Probes by Gold Nanoparticles Huixiang Li and Lewis J. Rothberg*
Department of Chemistry, University of Rochester, Rochester, New York 14627
Simple, fast, economical, and sensitive detection of specific DNA sequences is crucial to pathogen detection and biomedical research. We have designed a novel fluorescent assay for DNA hybridization based on the electrostatic properties of DNA. We exploit the ability to create conditions where single-stranded DNA adsorbs on negatively charged gold nanoparticles while double-stranded DNA does not. Dye-tagged probe sequences have their fluorescence efficiently quenched when they are mixed with gold nanoparticles unless they hybridize with components of the analyte. Subfemtomole amounts of untagged target are detected in minutes using commercially available materials. Target sequences in complex mixtures of DNA and single-base mismatches in DNA sequences are easily detected. Detection of specific oligonucleotide sequences is important for clinical diagnosis and biochemical research. Present assays are dominated by chip-based methodologies1,2 that have two principal disadvantages. First, target labeling is usually required, and second, hybridization to sterically constrained probes on surfaces is slow. Approaches such as sandwich assays,3-6 immobilized molecular beacons,7-9 surface plasmon resonance,10 porous silicon microcavity emission,11 and reflective interferometry12-14 avoid the former problem but still require complex * To whom correspondence should be addressed. E-mail: rothberg@ chem.rochester.edu. (1) Epstein, J. R.; Biran, I.; Walt, D. R. Anal. Chim. Acta 2002, 469, 3-36. (2) Chee, M.; Yang, R.; Hubbell, E.; Berno, A.; Huang, X. C.; Stern, D.; Winkler, J.; Lockhart, D. J.; Morris, M. S.; Fodor, S. P. A. Science 1996, 274, 610614. (3) Elghanian, R.; Storhoff, J. J.; Mucic, R. C.; Letsinger, R. L.; Mirkin, C. A. Science 1997, 277, 1078-1081. (4) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000, 289, 1757-1760. (5) Cao, Y. W. C.; Jin, R. C.; Mirkin, C. A. Science 2002, 297, 1536-1540. (6) Park, S. J.; Taton, T. A.; Mirkin, C. A. Science 2002, 295, 1503-1506. (7) Dubertret, B.; Calame, M.; Libchaber, A. J. Nat. Biotechnol. 2001, 19, 365370. (8) Maxwell, D. J.; Taylor, J. R.; Nie, S. J. Am. Chem. Soc. 2002, 124, 96069612. (9) Du, H.; Disney, M. D.; Miller, B. L.; Krauss, T. D. J. Am. Chem. Soc. 2003, 125, 4012-4013. (10) Brockman, J. M.; Nelson, B. P.; Corn, R. M. Annu. Rev. Phys. Chem. 2000, 51, 41-63. (11) Chan, S.; Li, Y.; Rothberg, L. J.; Miller, B. L.; Fauchet, P. M. Mater. Sci. Eng. C 2001, 15, 277-282. (12) Lin, V. S. Y.; Motesharei, K.; Dancil, K. P. S.; Sailor, M. J.; Ghadiri, M. R. Science 1997, 278, 840-843. (13) Pan, S.; Rothberg, L. J. Nano Lett. 2003, 3, 811-814.
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surface attachment chemistry for probe immobilization and suffer from slow response. In several of these cases, a nontrivial rinse step is required to remove unbound target or a second hybridization step is required in the assay. We report a simple, general method for sensitive detection of oligonucleotide sequences that solves these problems. The underlying principle derives from our recent observation that single- and double-stranded DNA (ds-DNA) have different propensity to adsorb to citrate-coated gold nanoparticles.15 In particular, single-stranded DNA (ss-DNA) sticks to these particles so that fluorescence from dye-tagged single-stranded probes is quenched unless they bind to target oligonucleotides. In this way, no target labeling is required and hybridization between probe and target is performed in solution under conditions that can be specified independent of the assay. Moreover, only commercially available materials are required and the entire assay takes less than 10 min. We show that it is also straightforward to detect single-base mismatches and to work in complex target mixtures using our approach. EXPERIMENTAL SECTION Materials. Hydrogen tetrachloroaurate(III) (HAuCl4‚3H2O), 99.99%, and sodium citrate (Na3C6H5O7‚2H2O), 99%, were purchased from Alfa Aesar and used without further purification. Potassium phosphate, monobasic, anhydrous 99.999%, and sodium phosphate, dibasic, anhydrous, 99.999%, were obtained from Aldrich Chemical and used as supplied. Sodium chloride crystals were purchased from Mallinckrodt. DNA oligonucleotides were synthesized and purified by MWG Biotech. The sequences of the oligomers used in this work are as follows: (1) rhodamine red-5′-AGG AAT TCC ATA GCT-3′ (probe I) and (2) its complement (c-target I); (3) 5′-TAA CAA TAA TCC CTC-3′ (nc-target I); (4) 5′- AC TAG GCA CTG TAC GCC AGC TAT GGA ATT CCT TAG CTA TGA GAT CCT TCG-3′ (mctarget I); (5) 5′-GT TAG CTA TGA GAT CCT TCG TAG GCA CTG TAC GCC AGC TAT GGA ATT CCT-3′ (ec-target I); (6) 5′TGT GTT GAA CCT GGT GAA GTT GTA ATC TGG AAC TTG TTG AGC AGA GGT TC-3′ (ncl-target I); (7) 5′- AC TAG GCA CTG TAC GCC AGC TAT CGA ATT CCT TAG CTA TGA GAT CCT TCG-3′ (mmc-target I); (8) 5′-GT TAG CTA TGA GAT CCT (14) Lu, J.; Strohsahl, C. M.; Miller, B. L.; Rothberg, L. J. Anal. Chem., in press. (15) Li, H. X.; Rothberg, L. J. Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold particles. Proc. Natl. Acad. Sci. U.S.A., in press. 10.1021/ac049173n CCC: $27.50
© 2004 American Chemical Society Published on Web 08/14/2004
Figure 1. Preferential quenching of tagged ss-DNA fluorescence by gold nanoparticles. (A) Fluorescence spectra of mixtures of 5 µL (10 µM) of trial hybridization solutions of probe I and c-target I (solid squares) or probe I and nc-target I (open squares) with 500 µL of gold colloid and 500 µL of 10 mM PBS containing 0.1 M NaCl. (B) The fluorescence spatial intensity profile of a 2-µL aliquot of assay solution measured with a confocal fluorescence microscope. The assay solution contains 0.5 µL (0.1 µM) of the trial hybridization solution mixed with 500 µL of gold colloid diluted with deionized water by a factor 20 and 500 µL of 10 mM PBS containing 0.1 M NaCl. Solid circles were recorded on a 2-µL aliquot from the mixture containing c-target I and the open circles on a 2-µL aliquot from the mixture containing nc-target I.
TCG TAG GCA CTG TAC GCC AGC TAT CGA ATT CCT-3′ (mectarget I); (9) Cy5-5′-TAG CTA TGG AAT TCC TCG TAG GCA3′ (probe II) and (10) (c-target II) its complement; (11) 5′-ATG GCA ACT ATA CGC GCT AC-3′ (nc-target II). The underlined portions of the sequences indicate positions along the target strands that are complementary to probe I. The italicized bases are the single bases in those sequences that are mismatched with probe I. Gold Nanoparticle Synthesis. Gold particles with 13-nm diameter were synthesized by reduction of HAuCl4.16 Trial Hybridization. Probes were oligonucleotide sequences complementary to a segment of the desired target and containing rhodamine red or Cy-5 attached at the 5′ end. These were hybridized with targets in 10 mM phosphate buffer solution (pH 7.0) with 0.3 M NaCl for 5 min at room temperature. Assay Protocol. An aliquot of the trial hybridization solution is 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 is added immediately afterward unless otherwise specified in the figure captions. Fluorescence Detection. The fluorescence of this mixture is recorded within 10 min using either a fluorometer or a fluorescence microscope and camera. Fluorescence spectra were measured on a fluorometer (Fluorolog 3, Jobin Yvon) with excitation at 570 nm and emission range from 585 to 680 nm. Spectrometer slits were set for 4-nm band-pass. Fluorescence images were recorded with an inverted confocal microscope (TE300, Nikon) equipped with a holographic notch filter to reject scattered excitation light and a narrow band-pass interference filter (585-nm center wavelength, 10-nm band-pass) in the fluorescence band of the tagged oligonucleotide. Fluorescence was excited by the 514-nm line of a continuous wave argon ion laser (∼100 µW) and detected with a photomultiplier. RESULTS AND DISCUSSION Differential Fluorescence Quenching of Dye-Tagged Single- and Double-Stranded DNA. DNA oligonucleotides labeled with rhodamine red fluorescent dye covalently attached at the 5′
end are used as probes. Several microliters of micromolar solutions of probe are exposed to the target sequences for trial hybridization in 10 mM phosphate buffer with 0.3 M NaCl. The hybridization solutions are added to colloidal gold suspensions, and additional phosphate buffer saline solution is added to assist in stabilizing ds-DNA against dehybridization in the colloid. Figure 1A illustrates the result of a measurement comparing the photoluminescence from trial solutions with complementary and noncomplementary targets. Fluorescence contrast greater than 100:1 is observed because unhybridized probes efficiently adsorb on the gold nanoparticles so that their fluorescence is quenched. The adsorption mechanism is entirely electrostatic and is discussed in more detail in our previous work.15 Briefly, the negatively charged phosphate backbone of the DNA is strongly repelled by the negative citrate ions coating the gold nanoparticles. While this largely prevents ds-DNA from adsorbing on the gold, this is not the case for ss-DNA even though it assumes a coiled geometry at ambient temperatures. When structural fluctuations of ss-DNA permit transient unraveling of sections of the oligonucleotide with their bases oriented toward the gold surface, an electrostatic attraction between the dipoles of the nanoparticle double layer and the ss-DNA double layer results. The adsorption and concomitant fluorescence quenching are irreversible. The differential fluorescence quenching in our approach is in some sense the inverse of the fluorescence resonance energy-transfer (FRET) method of Bazan and co-workers17 but related in that they both rely on electrostatics. In the present protocol, we quench unbound probe fluorescence by gold, while in that case, bound probe fluorescence is enhanced by conjugated polymer absorption and FRET. Much higher signal contrast between target and control analytes is obtained in the present work. Since relatively large volumes of solution are required for a typical fluorometer, it is not practical to assess the sensitivity of the method using that instrument. Figure 1B illustrates measuring the fluorescence of a very small aliquot of the trial hybridization solution containing only 0.1 fmol of target (a 2-µL aliquot of 0.5 µL of 0.1 µM DNA solution diluted in gold colloid and phosphate buffer to 1 mL), and this is easily detected with a fluorescence
(16) Gradar, K. C.; Freeman, R. G.; Hommer, M. B.; Natan, M. J. Anal. Chem. 1995, 67, 735-743.
(17) Gaylord, B. S.; Heeger, A. J.; Bazan, G. C. J. Am. Chem. Soc. 2003, 125, 896-900.
Analytical Chemistry, Vol. 76, No. 18, September 15, 2004
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Figure 2. Detection of a long target and a long target in a mixture. (A) Long oligonucleotide detection. Fluorescence spectra were recorded from solutions containing probe I and mc-target I (solid squares), probe I and ec-target I (open squares), and probe I and ncl-target I (solid triangles), respectively. The sample consists of 4 µL (10 µM) of trial hybridization solution, 500 µL of gold colloid, and 500 µL of 10 mM PBS containing 0.1 M NaCl. (B) Long target detection in a mixture. Fluorescence spectra were recorded from mixtures containing 1% as much mc-target I (solid squares) and 1% as much ec-target I (open squares) as ncl-target I (solid triangles). The amounts of the oligonucleotides in the trial hybridization solution are 10 pmol of nc-target, 0.5 pmol of probe, and 0.1 pmol each of mc-target I and ec-target I in 0.5 µL. These are added to 500 µL of gold colloid (diluted with 250 µL of water) and then 500 µL of 10 mM PBS containing 0.1 M NaCl is added.
microscope and camera. Since the method is essentially a null experiment in that no fluorescence is observed when there is no target present, the sensitivity can be improved by optimization of the light collection. Application to Long Target Sequences. 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 polymerase chain reaction (PCR). Figure 2A depicts the results of proof-of-principle experiments for detecting matches to sequences on long targets. Although large portions of the target remain single stranded and will presumably have the electrostatic properties of ss-DNA, we have no difficulty using the assay to determine whether these long targets contain sequences complementary to our short dye-tagged probes. The reason we do not observe adsorption and quenching in this case is that long ss-DNA sequences adsorb on the gold nanoparticles at a much slower rate, an observation we have documented elsewhere.18 Thus, our technique is most practical when short dye-tagged probes (
