An interfacial capacitance measurement electrochemical technique has been used for the sensing of self-assembled DNA hairpin probes (M. tuberculosis and B. anthracis) attached to Au electrodes. The double-layer capacitance ( ) was determined with electrochemical perturbations from 0.2?V to 0.5?V versus Ag/AgCl at a Au/M. tuberculosis DNA hairpin probe at surface coverage Au electrodes. The capacitance study was done at pH 7, which was necessary to maintain the M. tuberculosis and B. anthracis DNA probes closed during the electrochemical perturbation. Detailed experimental analysis carried out by repetitively switching the electrochemical potential between 0.2 and 0.5?V (versus Ag/AgCl) strongly supports the use of capacitance measurements as a tool to detect the hybridization of DNA targets. A large change in the capacitance deference between 0.2 and 0.5?V was observed in the DNA hybridization process. Therefore, no fluorophores or secondary transducers were necessary to sense a DNA target for both DNA hairpins. 1. Introduction The behavior of DNA attached onto metallic and nonmetallic surfaces via self-assembly with various chemistries (e.g., Au-S) may have applications in biomedical devices. For example, single-stranded DNA (ssDNA) self-assembled on a metallic interface such as gold [1, 2] or on nonmetals such as carbon nanotubes [3] and diamond [4–6] has potential use in DNA microarrays [7]. In addition, detection of DNA hybridization has been possible with techniques using different types of reporting, including fluorescence [8–11], chronocoulometry [12–14], surface plasmon resonance (SPR) [15, 16], colloidal labeling [17–19], and polarization modulation infrared reflection absorption spectroscopy [20]. Electroactive molecules can also be used to monitor the electron transfer mechanism during the hybridization process [21]. Here, we present a nonfaradaic electrochemical method based on capacitive measurement to sense DNA hairpin modification and hybridization. A nucleic acid probe has been developed to recognize specific DNA targets in solution [22]. These probes, called molecular beacons, are DNA hairpins with a fluorophore-quencher pair, which is completely unable to fluoresce when the two components are in close proximity (i.e., closed molecular beacon). When the molecular beacon spontaneously changes its conformation (like during hybridization), the fluorophore attached to one end of the molecule is no longer quenched as the quencher moves away. The capacity of this DNA hairpin has shown to discriminate between alleles with high specificity when
References
[1]
T. M. Herne and M. J. Tarlov, “Characterization of DNA probes immobilized on gold surfaces,” Journal of the American Chemical Society, vol. 119, no. 38, pp. 8916–8920, 1997.
[2]
S. O. Kelley, J. K. Barton, N. M. Jackson et al., “Orienting DNA helices on gold using applied electric fields,” Langmuir, vol. 14, no. 24, pp. 6781–6784, 1998.
[3]
C. S. Lee, S. E. Baker, M. S. Marcus, W. Yang, M. A. Eriksson, and R. J. Hamers, “Electrically addressable biomolecular functionalization of carbon nanotube and carbon nanofiber electrodes,” Nano Letters, vol. 4, no. 9, pp. 1713–1716, 2004.
[4]
W. Yang, O. Auciello, J. E. Butler et al., “DNA-modified nanocrystalline diamond thin-films as stable, biologically active substrates,” Nature Materials, vol. 1, no. 4, pp. 253–257, 2002.
[5]
T. Knickerbocker, T. Strother, M. P. Schwartz et al., “DNA-modified diamond surfaces,” Langmuir, vol. 19, no. 6, pp. 1938–1942, 2003.
[6]
W. Yang, J. E. Butler, J. N. Russell Jr., and R. J. Hamers, “Interfacial electrical properties of DNA-modified diamond thin films: intrinsic response and hybridization-induced field effects,” Langmuir, vol. 20, no. 16, pp. 6778–6787, 2004.
[7]
S. P. A. Fodor, “Massively parallel genomics,” Science, vol. 277, no. 5324, pp. 393–395, 1997.
[8]
S. P. A. Fodor, J. L. Read, M. C. Pirrung, L. Stryer, A. T. Lu, and D. Solas, “Light-directed, spatially addressable parallel chemical synthesis,” Science, vol. 251, no. 4995, pp. 767–773, 1991.
[9]
D. J. Lockhart and E. A. Winzeler, “Genomics, gene expression and DNA arrays,” Nature, vol. 405, no. 6788, pp. 827–836, 2000.
[10]
M. L. Bulyk, E. Gentalen, D. J. Lockhart, and G. M. Church, “Quantifying DNA-protein interactions by double-stranded DNA arrays,” Nature Biotechnology, vol. 17, no. 6, pp. 573–577, 1999.
[11]
M. Chee, R. Yang, E. Hubbell et al., “Accessing genetic information with high-density DNA arrays,” Science, vol. 274, no. 5287, pp. 610–614, 1996.
[12]
M. B. Esch, L. E. Locascio, M. J. Tarlov, and R. A. Durst, “Detection of viable Cryptosporidium parvum using DNA-modified liposomes in a microfluidic chip,” Analytical Chemistry, vol. 73, no. 13, pp. 2952–2958, 2001.
[13]
A. B. Steel, T. M. Herne, and M. J. Tarlov, “Electrochemical quantitation of DNA immobilized on gold,” Analytical Chemistry, vol. 70, no. 22, pp. 4670–4677, 1998.
[14]
A. B. Steel, T. M. Herne, and M. J. Tarlov, “Electrostatic interactions of redox cations with surface-immobilized and solution DNA,” Bioconjugate Chemistry, vol. 10, no. 3, pp. 419–423, 1999.
[15]
L. He, M. D. Musick, S. R. Nicewarner et al., “Colloidal Au-enhanced surface plasmon resonance for ultrasensitive detection of DNA hybridization,” Journal of the American Chemical Society, vol. 122, no. 38, pp. 9071–9077, 2000.
[16]
J. M. Brockman, A. G. Frutos, and R. M. Corn, “A multistep chemical modification procedure to create dna arrays on gold surfaces for the study of protein-DNA interactions with surface plasmon resonance imaging,” Journal of the American Chemical Society, vol. 121, no. 35, pp. 8044–8051, 1999.
[17]
M. L. Sauthier, R. Lloyd Carroll, C. B. Gorman, and S. Franzen, “Nanoparticle layers assembled through DNA hybridization: characterization and optimization,” Langmuir, vol. 18, no. 5, pp. 1825–1830, 2002.
[18]
Y. Cao, R. Jin, and C. A. Mirkin, “DNA-modified core-shell Ag/Au nanoparticles,” Journal of the American Chemical Society, vol. 123, no. 32, pp. 7961–7962, 2001.
[19]
L. M. Demers, C. A. Mirkin, R. C. Mucic et al., “A fluorescence-based method for determining the surface coverage and hybridization efficiency of thiol-capped oligonucleotides bound to gold thin films and nanoparticles,” Analytical Chemistry, vol. 72, no. 22, pp. 5535–5541, 2000.
[20]
S. H. Brewer, S. J. Anthireya, S. E. Lappi, D. L. Drapcho, and S. Franzen, “Detection of DNA hybridization on gold surfaces by polarization modulation infrared reflection absorption spectroscopy,” Langmuir, vol. 18, no. 11, pp. 4460–4464, 2002.
[21]
C. Fan, K. W. Plaxco, and A. J. Heeger, “Electrochemical interrogation of conformational changes as a reagentless method for the sequence-specific detection of DNA,” Proceedings of the National Academy of Sciences of the United States of America, vol. 100, no. 16, pp. 9134–9137, 2003.
[22]
S. Tyagi and F. R. Kramer, “Molecular beacons: probes that fluoresce upon hybridization,” Nature Biotechnology, vol. 14, no. 3, pp. 303–308, 1996.
[23]
S. Tyagi, D. P. Bratu, and F. R. Kramer, “Multicolor molecular beacons for allele discrimination,” Nature Biotechnology, vol. 16, no. 1, pp. 49–53, 1998.
[24]
L. G. Kostrikis, S. Tyagi, M. M. Mhlanga, D. D. Ho, and F. R. Kramer, “Spectral genotyping of human alleles,” Science, vol. 279, no. 5354, pp. 1228–1229, 1998.
[25]
S. A. E. Marras, F. Russell Kramer, and S. Tyagi, “Multiplex detection of single-nucleotide variations using molecular beacons,” Genetic Analysis—Biomolecular Engineering, vol. 14, no. 5-6, pp. 151–156, 1999.
[26]
G. Bonnet, S. Tyagi, A. Libchaber, and F. R. Kramer, “Thermodynamic basis of the enhanced specificity of structured DNA probes,” Proceedings of the National Academy of Sciences of the United States of America, vol. 96, no. 11, pp. 6171–6176, 1999.
[27]
R. Bar-Ziv and A. Libchaber, “Effects of DNA sequence and structure on binding of RecA to single-stranded DNA,” Proceedings of the National Academy of Sciences of the United States of America, vol. 98, no. 16, pp. 9068–9073, 2001.
[28]
Y. Gao, L. K. Wolf, and R. M. Georgiadis, “Secondary structure effects on DNA hybridization kinetics: a solution versus surface comparison,” Nucleic Acids Research, vol. 34, no. 11, pp. 3370–3377, 2006.
[29]
G. Liu, Y. Wan, V. Gau et al., “An enzyme-based E-DNA sensor for sequence-specific detection of femtomolar DNA targets,” Journal of the American Chemical Society, vol. 130, no. 21, pp. 6820–6825, 2008.
[30]
S. Song, Z. Liang, J. Zhang, L. Wang, G. Li, and C. Fan, “Gold-nanoparticle-based multicolor nanobeacons for sequence-specific DNA analysis,” Angewandte Chemie—International Edition, vol. 48, no. 46, pp. 8670–8674, 2009.
[31]
J. Rivera-Gandía and C. R. Cabrera, “Self-assembled monolayers of 6-mercapto-1-hexanol and mercapto-n-hexyl-poly(dT)18-fluorescein on polycrystalline gold surfaces: an electrochemical impedance spectroscopy study,” Journal of Electroanalytical Chemistry, vol. 605, no. 2, pp. 145–150, 2007.
[32]
W. Cai, J. R. Peck, D. W. van der Weide, and R. J. Hamers, “Direct electrical detection of hybridization at DNA-modified silicon surfaces,” Biosensors and Bioelectronics, vol. 19, no. 9, pp. 1013–1019, 2004.
[33]
B. C. Jacquot, N. Mu?oz, D. W. Branch, and E. C. Kan, “Non-Faradaic electrochemical detection of protein interactions by integrated neuromorphic CMOS sensors,” Biosensors and Bioelectronics, vol. 23, no. 10, pp. 1503–1511, 2008.
[34]
E. Katz and I. Willner, “Probing biomolecular interactions at conductive and semiconductive surfaces by impedance spectroscopy: routes to impedimetric immunosensors, DNA-sensors, and enzyme biosensors,” Electroanalysis, vol. 15, no. 11, pp. 913–947, 2003.
[35]
E. Sharon, R. Freeman, T. V. Ran, and I. Willner, “Impedimetric or ion-sensitive field-effect transistor (ISFET) aptasensors based on the self-assembly of au nanoparticle-functionalized supramolecular aptamer nanostructures,” Electroanalysis, vol. 21, no. 11, pp. 1291–1296, 2009.
[36]
J. Wang, “Towards genoelectronics: electrochemical biosensing of DNA hybridization,” Chemistry-A European Journal, vol. 5, p. 1681, 1999.
[37]
S. R. Mikklessen, “Electrochecmical biosensors for DNA sequence detection,” Electroanalysis, vol. 8, p. 15, 1996.
[38]
E. Pale?ek and M. Fojta, “Detecting DNA hybridization and damage,” Analytical Chemistry, vol. 73, no. 3, 2001.
[39]
H. Aoki, P. Buhlmann, and Y. Umezawa, “Electrochemical detection of a one-base mismatch in an oligonucleotide using ion-channel sensors with self-assembled PNA monolayers,” Electroanalysis, vol. 12, no. 16, pp. 1272–1276, 2000.
[40]
A. J. Bard and L. R. Faulkner, Electrochemical Methods: Fundamentals and Applications, chapter 10, 2nd edition, 2001.
[41]
C. Xi, M. Balberg, S. A. Boppart, and L. Raskin, “Use of DNA and peptide nucleic acid molecular beacons for detection and quantification of rRNA in solution and in whole cells,” Applied and Environmental Microbiology, vol. 69, no. 9, pp. 5673–5678, 2003.
[42]
X. Liu and W. Tan, “A fiber-optic evanescent wave DNA biosensor based on novel molecular beacons,” Analytical Chemistry, vol. 71, p. 5054, 1999.