Anal. Chem. 2005, 77, 7810-7815
Detection of Human Immunodeficiency Virus Type 1 DNA Sequence Using Plasmonics Nanoprobes Musundi B. Wabuyele and Tuan Vo-Dinh*
Center for Advanced Biomedical Photonics, Oak Ridge National Laboratory, P.O. Box 2008, Oak Ridge, Tennnessee 37831
This paper describes the use of plasmonics-based nanoprobes that act as molecular sentinels for DNA diagnostics. The plasmonics nanoprobe comprises a metal nanoparticle and a stem-loop DNA molecule tagged with a Raman label. The nanoprobe utilizes the specificity and selectivity of the DNA hairpin probe sequence to detect a specific target DNA sequence of interest. In the absence of target DNA, the stem-loop configuration maintains the Raman label in proximity to the metal nanoparticle, inducing an intense surface-enhanced Raman scattering (SERS) effect that produces a strong Raman signal upon laser excitation. Upon hybridization of a complementary target DNA sequence to the nanoprobe, the stem-loop configuration is disrupted, causing the Raman label to physically separate from the metal nanoparticle, thus quenching the SERS signal. The usefulness and potential application of the plasmonics nanoprobe for diagnosis is demonstrated using the gag gene sequence of the human immunodeficiency virus type 1 (HIV-1). We successfully demonstrated the specificity and selectivity of the plasmonics nanoprobes to detect PCR amplicons of the HIV gene. The potential for combining the spectral selectivity and high sensitivity of the SERS process with inherent molecular specificity of DNA hairpins to diagnose molecular target sequences in homogeneous solutions is discussed. The worldwide epidemiological data regarding the autoimmune deficiency syndrome (AIDS) pandemic indicate a clear need for researchers to develop rapid and sensitive molecular screening and diagnostic techniques that detect the virus at the earliest possible time following infection.1,2 Therapies using triple combinations of antiretroviral agents increased the need of early detection and ultrasensitive assays for human immunodeficiency virus type 1 (HIV-1).3-6 The virus should be detected before it * Corresponding author. Phone: (865) 574-6249. Fax: (865) 576-7651. E-mail:
[email protected]. (1) UNAIDS/World Health Organization. AIDS epidemic update 2001. Joint United Nations Programme on HIV/AIDS-World Health Organization, Geneva, Switzerland, 2001. (2) Schwartz, S. A.; Nair, M. P. N. Clin. Diagn. Lab. Immunol. 1999, 6, 295305. (3) Miles, S. A.; Balden, E.; Magpantay, L.; Wei, L.; Leiblein, A.; Hofheinz, D.; Toedter, G.; Stiehm, E. R.; Bryson, Y. N. Engl. J. Med., 1993, 328, 297302. (4) Larder, B. A.; Kemp, S. D. Science 1989, 246, 1155-1158. (5) Mellors, J. W.; Rinaldo, C. R.; Gupta, P.; White, R. M.; Todd, J. A.; Kingsley, L. A. Science 1996, 272, 1167-1170.
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destroys the immune system. Serological tests such as the enzyme-linked immunosorbent assay (ELISA), particle agglutination assay, and Western blot (WB) assay for the detection of HIV antibodies are routinely utilized for the screening and confirmation of HIV infection.7,8 Although ELISA and WB assay are very sensitive, they require relatively complex instrumentation and elaborate sample preparation. There is, therefore, a critical need to develop alternate detection methods that are rapid and offer comparable sensitivity and molecular specificity and could be performed using the relatively simple homogeneous assay format. Reliable detection of the retroviral DNA has been obtained with polymerase chain reaction (PCR) assays.9,10 PCR techniques allow the detection of actual nucleic acid sequences that are part of the HIV-1 virus. This technique generates millions of copies of target DNA sequences and multiple virus-specific sequences can be searched for and amplified in one reaction assay. Although PCR processes are entirely automated, detection and quantification of the product remains an area of interest to most researchers. Several fluorescence resonance energy-transfer-based homogeneous genotyping assays, which apply hybridization techniques, have been developed.11-15 Nevertheless, the need for alternative, rapid, and selective assays has continued to encourage researchers to explore other optical detection technologies for diagnostic application having sensitivity comparable to that of fluorescence but having unique and complementary advantages.16-18 Raman spectroscopy is an important analytical technique for chemical (6) O’Brien, T.; Blattner, W.; Waters, D.; Eyster, E.; Hilgartner, M. W.; Cohen, A. R.; Luban, N.; Hatzakis, A.; Aledort, L. M.; Rosenberg, P. S.; Miley, W. J.; Kroner, B. L.; Goedert, J. J. J. Am. Med. Assoc. 1996, 276, 105-110. (7) Pantaleo, G.; Graziosi, C.; Fauci, A. S. N. Engl. J. Med. 1993, 328, 327335. (8) Sutthent, R.; Gaudart, N.; Chokpaibulkit, K.; Tanliang, N.; Kanoksinsombath, C.; Chaisilwatana, P. J. Clin. Microbiol. 2003, 41, 1016-1022. (9) Palmer, S.; Wiegand, A. P.; Maldarelli, F.; Bazmi, H.; Mican, J. M.; Polis, M.; Dewar, R. L.; Planta, A.; Liu, S.; Metcalf, J. A.; Mellors, J. W.; Coffin, J. M. J. Clin. Microbiol. 2003, 41, 4531-4536. (10) Mulder, J.; McKinney, N.; Christopherson, C.; Sninsky, J.; Greenfield, L.; Kwok, S. J. Clin. Microbiol. 1994, 32, 292-300. (11) Vet, J. A.; Majithia, A. R.; Marras, S. A.; Tyagi, S.; Dube, S.; Poiesz, B. J.; Kramer, F. R. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 6394-6399. (12) Livak, K. J.; Flood, S. J. A.; Marmaro, J.; Giusti, W.; Deetz, K. PCR Methods Appl. 1995, 4, 1-6. (13) Leone, G.; van Schijndel, H.; van Gemen, B.; Kramer, F. R.; Schoen, C. D. Nucleic Acids Res. 1998, 26, 2150-2155. (14) Thelwell, N.; Millington, S. Solinas, A.; Booth, J.; Brown, T. Nucleic Acids Res. 2000, 28, 3752-3761. (15) Tyagi, S., Kramer, F. R. Nat. Biotechnol. 1996, 14, 303-308. (16) Graham, D.; Mallinder, B. J.; Smith, E. W. Angew. Chem., Int. Ed. 2000, 39, 1061-1063. (17) Cao, Y. C.; Jin, R.; Mirkin, C. A. Science 2002, 297, 1536-1540. 10.1021/ac0514671 CCC: $30.25
© 2005 American Chemical Society Published on Web 10/12/2005
and biological analysis due to the wealth of information on molecular structures, surface processes, and interface reactions that can be extracted from experimental data. The spectral selectivity associated with the narrow emission lines and the molecular specific vibrational bands of Raman labels make it an ideal tool for molecular genotyping. Fluorescence spectra are relatively broad (typically 50-100 nm, half-widths) and lead easily to spectral overlap when multiple probes are used simultaneously. Raman spectra of the same dye are much narrower (5 min) and surface modification using alkanethiols or thiolated poly(ethylene glycol)s should improve the DNA binding kinetics at metal surfaces.25,34,35 This result demonstrates that MS technology is a promising diagnostic tool that can be applied to real-time detection of DNA targets in solution. CONCLUSION We have demonstrated the feasibility and usefulness of a new type of plasmonics-based gene probe to detect nucleic acid sequences associated with the HIV-1 gene. The results of this study indicate that the SERS MS approach, which involves homogeneous assays, greatly simplifies experimental procedures and could potentially be used in assays that require rapid, highvolume identification of genomic materials. The possibility for single-molecule detection using colloidal SERS has been previously demonstrated by our group and many others.21-23 The intrinsic Raman enhancement factors (order of ∼1014-1015) have enabled the sensitivity of SERS to be comparable to that of fluorescence. However, because fluorescence spectra have relatively broad band features, spectral overlap is a limitation for the use of a large number of fluorescence labels simultaneously. The (35) Jin, R.; Wu, G.; Li, Z.; Mirkin, C. A.; Schatz, G. C. J. Am. Chem. Soc. 2003, 125, 1643-1654.
highly specific and narrow Raman spectral bands minimize spectral overlap of different labels, thus offering greater multiplexing capabilities for detection. Therefore, the use of SERS plasmonics probes for detection has potential advantage over fluorescence probes. In addition to the advantage of a single excitation source, the nonresonant process observed in SERS allows the effect to be insensitive to photobleaching and self-quenching of optical tags. Finally, the innovative approach of combining plasmonics with DNA hairpin probe detection makes the technique more sensitive and selective and, thus, could provide a useful tool for molecular diagnostics. Due to these unique properties, the SERS molecular sentinel approach could contribute to the development of the next generation of DNA diagnostic tools for molecular screening and molecular imaging applications. ACKNOWLEDGMENT This work was sponsored by the Office of Biological and Environmental Research, U.S. Department of Energy, under Contract DE-AC05-00OR22725 with UT-Battelle, LLC; and we thank the Oak Ridge National Laboratory LDRD Program (Advanced Plasmonics Sensors) and the National Institute of Health for the support of this research. This research was also supported in part by the appointment of M.B.W. to the U.S. Department of Energy Laboratory Cooperative Postdoctoral Research Training Program administered by Oak Ridge Institute of Science and Education. SUPPORTING INFORMATION AVAILABLE Computer-generated HIV-1 MS, SK19 MS DNA hairpin structures, Figure S3, and Table S1 showing the various DNA probe sequences used. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review August 15, 2005. Accepted September 14, 2005. AC0514671
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