Colorimetric and Fluorescent Biosensors Based on Directed Assembly of Nanomaterials with Functional DNA

  • Juewen Liu
  • Yi Lu
Part of the Integrated Analytical Systems book series (ANASYS)


This chapter reviews recent progress in the interface between functional nucleic acids and nanoscale science and technology, and its analytical applications. In particular, the use of metallic nanoparticles as the color reporting groups for the action (binding, catalysis, or both) of aptamers, DNAzymes, and aptazymes is described in detail. Because metallic nanoparticles possess high extinction coefficients and distance-dependent optical properties, they allow highly sensitive detections with minimal consumption of materials. The combination of quantum dots (QDs) with functional nucleic acids as fluorescent sensors is also described. The chapter starts with the design of colorimetric and fluorescent sensors responsive to single analytes, followed by sensors responsive to multiple analytes with controllable cooperativity and multiplex detection using both colorimetric and fluorescent signals in one pot, and ends by transferring solution-based detections into litmus paper type of tests, making them generally applicable and usable for a wide range of on-site and real-time analytical applications such as household tests, environmental monitoring, and clinical diagnostics.


Colorimetric Sensor Organic Fluorophores Lateral Flow Device Thrombin Aptamer Functional Nucleic Acid 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank other Lu group members for helpful discussions and Ms. Natasha Yeung for proofreading the chapter. The Lu group research described in this chapter has been generously supported by the U.S. Department of Energy, National Science Foundation, Department of Defense, Department of Housing and Urban Development, and National Institute of Health.


  1. 1.
    Kruger, K., Grabowski, P.J., Zaug, A.J., Sands, J., Gottschling, D.E. and Cech, T.R. (1982) Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of tetrahymena. Cell 31:147–157.CrossRefGoogle Scholar
  2. 2.
    Guerrier-Takada, C., Gardiner, K., Marsh, T., Pace, N. and Altman, S. (1983) The RNA moiety of ribonuclease p is the catalytic subunit of the enzyme. Cell 35:849–857.CrossRefGoogle Scholar
  3. 3.
    Winkler, W., Nahvi, A. and Breaker, R.R. (2002) Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression. Nature (Lond.) 419:952–956.CrossRefGoogle Scholar
  4. 4.
    Breaker, R.R. (2004) Natural and engineered nucleic acids as tools to explore biology. Nature (Lond.) 432:838.CrossRefGoogle Scholar
  5. 5.
    Breaker, R.R. and Joyce, G.F. (1994) A DNA enzyme that cleaves RNA. Chem. Biol. 1: 223–229CrossRefGoogle Scholar
  6. 6.
    Tuerk, C. and Gold, L. (1990) Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage t4 DNA polymerase. Science 249:505–510.CrossRefGoogle Scholar
  7. 7.
    Ellington, A.D. and Szostak, J.W. (1990) In vitro selection of RNA molecules that bind specific ligands. Nature (Lond.) 346:818–822.CrossRefGoogle Scholar
  8. 8.
    Wilson, D.S. and Szostak, J.W. (1999) In vitro selection of functional nucleic acids. Annu. Rev. Biochem. 68:611–647.CrossRefGoogle Scholar
  9. 9.
    Jayasena, S.D. (1999) Aptamers: an emerging class of molecules that rival antibodies in diagnostics. Clin. Chem. 45:1628–1650.Google Scholar
  10. 10.
    Tang, J. and Breaker, R.R. (1997) Rational design of allosteric ribozymes. Chem. Biol. 4: 453–459.CrossRefGoogle Scholar
  11. 11.
    Lu, Y. (2002) New transition metal-dependent DNAzymes as efficient endonucleases and as selective metal biosensors. Chem. Eur. J. 8:4588–4596.CrossRefGoogle Scholar
  12. 12.
    Osborne, S.E. and Ellington, A.D. (1997) Nucleic acid selection and the challenge of combinatorial chemistry. Chem. Rev. 97:349–370.CrossRefGoogle Scholar
  13. 13.
    Breaker, R.R. (1997) In vitro selection of catalytic polynucleotides. Chem. Rev. 97:371–390.CrossRefGoogle Scholar
  14. 14.
    Sen, D. and Geyer, C.R. (1998) DNA enzymes. Curr. Opin. Chem. Biol. 2:680–687.CrossRefGoogle Scholar
  15. 15.
    Famulok, M. and Jenne, A. (1999) Catalysis based on nucleic acid structures. Top. Curr. Chem. 202:101–131.CrossRefGoogle Scholar
  16. 16.
    Joyce, G.F. (2004) Directed evolution of nucleic acid enzymes. Annu. Rev. Biochem. 73: 791–836.CrossRefGoogle Scholar
  17. 17.
    Achenbach, J.C., Chiuman, W., Cruz, R.P.G. and Li, Y. (2004) DNAzymes: from creation in vitro to application in vivo. Curr. Pharm. Biotechnol. 5:312–336.CrossRefGoogle Scholar
  18. 18.
    Silverman, S.K. (2005) In vitro selection, characterization, and application of deoxyribozymes that cleave RNA. Nucleic Acids Res. 33:6151–6163.CrossRefGoogle Scholar
  19. 19.
    Bruesehoff, P.J., Li, J., Augustine, A.J. and Lu, Y. (2002) Improving metal ion specificity during in vitro selection of catalytic DNA. Comb. Chem. High Throughput Screen. 5:327–335.Google Scholar
  20. 20.
    Stojanovic, M.N. and Landry, D.W. (2002) Aptamer-based colorimetric probe for cocaine. J. Am. Chem. Soc. 124:9678–9679.CrossRefGoogle Scholar
  21. 21.
    Yguerabide, J. and Yguerabide, E.E. (1998) Light-scattering submicroscopic particles as highly fluorescent analogs and their use as tracer labels in clinical and biological applications. I. Theory. Anal. Biochem. 262:137–156.CrossRefGoogle Scholar
  22. 22.
    Bohren, C.F. and Huffman, D.R. (1993) Absorption and scattering of light by small particles. Wiley, New York.Google Scholar
  23. 23.
    Jin, R., Wu, G., Li, Z., Mirkin, C.A. and Schatz, G.C. (2003) What controls the melting properties of DNA-linked gold nanoparticle assemblies? J. Am. Chem. Soc. 125:1643–1654.CrossRefGoogle Scholar
  24. 24.
    Mirkin, C.A., Letsinger, R.L., Mucic, R.C. and Storhoff, J.J. (1996) A DNA-based method for rationally assembling nanoparticles into macroscopic materials. Nature (Lond.) 382:607–609.CrossRefGoogle Scholar
  25. 25.
    Elghanian, R., Storhoff, J.J., Mucic, R.C., Letsinger, R.L. and Mirkin, C.A. (1997) Selective colorimetric detection of polynucleotides based on the distance-dependent optical properties of gold nanoparticles. Science 277:1078–1080.CrossRefGoogle Scholar
  26. 26.
    Rosi, N.L. and Mirkin, C.A. (2005) Nanostructures in biodiagnostics. Chem. Rev. 105: 1547–1562.CrossRefGoogle Scholar
  27. 27.
    Han, M., Gao, X., Su, J.Z. and Nie, S. (2001) Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nat. Biotechnol. 19:631–635.CrossRefGoogle Scholar
  28. 28.
    Alivisatos, A.P., Gu, W. and Larabell, C. (2005) Quantum dots as cellular probes. Annu. Rev. Biomed. Eng. 7:55–76.CrossRefGoogle Scholar
  29. 29.
    Medintz, I.L., Uyeda, H.T., Goldman, E.R. and Mattoussi, H. (2005) Quantum dot bioconju-gates for imaging, labelling and sensing. Nat. Mater. 4:435–446.CrossRefGoogle Scholar
  30. 30.
    Li, J., Zheng, W., Kwon, A.H. and Lu, Y. (2000) In vitro selection and characterization of a highly efficient Zn(ii)-dependent RNA-cleaving deoxyribozyme. Nucleic Acids Res. 28: 481–488.CrossRefGoogle Scholar
  31. 31.
    Santoro, S.W., Joyce, G.F., Sakthivel, K., Gramatikova, S. and Barbas, C.F., III. (2000) RNA cleavage by a DNA enzyme with extended chemical functionality. J. Am. Chem. Soc. 122: 2433–2439.CrossRefGoogle Scholar
  32. 32.
    Carmi, N., Shultz, L.A. and Breaker, R.R. (1996) In vitro selection of self-cleaving DNAs. Chem. Biol. 3:1039–1046.CrossRefGoogle Scholar
  33. 33.
    Carmi, N., Balkhi, H.R. and Breaker, R.R. (1998) Cleaving DNA with DNA. Proc. Natl. Acad. Sci. USA 95:2233–2237.CrossRefGoogle Scholar
  34. 34.
    Mei, S.H.J., Liu, Z., Brennan, J.D. and Li, Y. (2003) An efficient RNA-cleaving DNA enzyme that synchronizes catalysis with fluorescence signaling. J. Am. Chem. Soc. 125:412–420.CrossRefGoogle Scholar
  35. 35.
    Liu, J., Brown, A.K., Meng, X., Cropek, D.M., Istok, J.D., Watson, D.B. and Lu, Y. (2007) A catalytic beacon sensor for uranium with parts-per-trillion sensitivity and million-fold selectivity. Proc. Natl. Acad. Sci. USA 104:2056.CrossRefGoogle Scholar
  36. 36.
    Brown, A.K., Li, J., Pavot, C.M.B. and Lu, Y. (2003) A lead-dependent DNAzyme with a two-step mechanism. Biochemistry 42:7152–7161.CrossRefGoogle Scholar
  37. 37.
    Liu, J. and Lu, Y. (2003) A colorimetric lead biosensor using DNAzyme-directed assembly of gold nanoparticles. J. Am. Chem. Soc. 125:6642–6643.CrossRefGoogle Scholar
  38. 38.
    Liu, J. and Lu, Y. (2004) Optimization of a Pb2+-directed gold nanoparticle/DNAzyme assembly and its application as a colorimetric biosensor for Pb2+. Chem. Mater. 16:3231–3238.CrossRefGoogle Scholar
  39. 39.
    Liu, J. and Lu, Y. (2004) Accelerated color change of gold nanoparticles assembled by DNAzymes for simple and fast colorimetric Pb2+ detection. J. Am. Chem. Soc. 126: 12298–12305.CrossRefGoogle Scholar
  40. 40.
    Storhoff, J.J., Lazarides, A.A., Mucic, R.C., Mirkin, C.A., Letsinger, R.L. and Schatz, G.C. (2000) What controls the optical properties of DNA-linked gold nanoparticle assemblies? J. Am. Chem. Soc. 122:4640–4650.CrossRefGoogle Scholar
  41. 41.
    Liu, J. and Lu, Y. (2005) Stimuli-responsive disassembly of nanoparticle aggregates for light-up colorimetric sensing. J. Am. Chem. Soc. 127:12677–12683.CrossRefGoogle Scholar
  42. 42.
    Liu, J. and Lu, Y. (2006) Design of asymmetric DNAzymes for dynamic control of nanoparticle aggregation states in response to chemical stimuli. Org. Biomol. Chem. 4:3435–3441.CrossRefGoogle Scholar
  43. 43.
    Geyer, C.R. and Sen, D. (1997) Evidence for the metal-cofactor independence of an RNA phosphodiester-cleaving DNA enzyme. Chem. Biol. 4:579–593.CrossRefGoogle Scholar
  44. 44.
    Roth, A. and Breaker, R.R. (1998) An amino acid as a cofactor for a catalytic polynucleotide. Proc. Natl. Acad. Sci. USA 95:6027–6031.CrossRefGoogle Scholar
  45. 45.
    Breaker, R.R. (2002) Engineered allosteric ribozymes as biosensor components. Curr. Opin. Biotechnol. 13:31–39.CrossRefGoogle Scholar
  46. 46.
    Wang, D.Y., Lai, B.H.Y. and Sen, D. (2002) A general strategy for effector-mediated control of RNA-cleaving ribozymes and DNA enzymes. J. Mol. Biol. 318:33–43.CrossRefGoogle Scholar
  47. 47.
    Huizenga, D.E. and Szostak, J.W. (1995) A DNA aptamer that binds adenosine and ATP. Biochemistry 34:656–665.CrossRefGoogle Scholar
  48. 48.
    Liu, J. and Lu, Y. (2004) Adenosine-dependent assembly of aptazyme-functionalized gold nanoparticles and its application as a colorimetric biosensor. Anal. Chem. 76:1627–1632.CrossRefGoogle Scholar
  49. 49.
    Ho, H.-A. and Leclerc, M. (2004) Optical sensors based on hybrid aptamer/conjugated polymer complexes. J. Am. Chem. Soc. 126:1384–1387.CrossRefGoogle Scholar
  50. 50.
    Ho, H.-A., Bera-Aberem, M. and Leclerc, M. (2005) Optical sensors based on hybrid DNA/ conjugated polymer complexes. Chem. Eur. J 11:1718–1724.CrossRefGoogle Scholar
  51. 51.
    Dore, K., Dubus, S., Ho, H.-A., Levesque, I., Brunette, M., Corbeil, G., Boissinot, M., Boivin, G., Bergeron, M.G., Boudreau, D. and Leclerc, M. (2004) Fluorescent polymeric transducer for the rapid, simple, and specific detection of nucleic acids at the zeptomole level. J. Am. Chem. Soc. 126:4240–4244.CrossRefGoogle Scholar
  52. 52.
    Pavlov, V., Xiao, Y., Shlyahovsky, B. and Willner, I. (2004) Aptamer-functionalized Au nanopar-ticles for the amplified optical detection of thrombin. J. Am. Chem. Soc. 126:11768–11769.CrossRefGoogle Scholar
  53. 53.
    Padmanabhan, K., Padmanabhan, K.P., Ferrara, J.D., Sadler, J.E. and Tulinsky, A. (1993) The structure of a-thrombin inhibited by a 15-mer single-stranded DNA aptamer. J. Biol. Chem. 268:17651–17654.Google Scholar
  54. 54.
    Huang, C.-C., Huang, Y.-F., Cao, Z., Tan, W. and Chang, H.-T. (2005) Aptamer-modified gold nanoparticles for colorimetric determination of platelet-derived growth factors and their receptors. Anal. Chem. 77:5735–5741.CrossRefGoogle Scholar
  55. 55.
    Liu, J. and Lu, Y. (2006) Fast colorimetric sensing of adenosine and cocaine based on a general sensor design involving aptamers and nanoparticles. Angew. Chem. Int. Ed. 45:90–94.CrossRefGoogle Scholar
  56. 56.
    Nutiu, R. and Li, Y. (2003) Structure-switching signaling aptamers. J. Am. Chem. Soc. 125: 4771–4778.CrossRefGoogle Scholar
  57. 57.
    Nutiu, R., Mei, S., Liu, Z. and Li, Y. (2004) Engineering DNA aptamers and DNA enzymes with fluorescence-signaling properties. Pure Appl. Chem. 76:1547–1561.CrossRefGoogle Scholar
  58. 58.
    Nutiu, R. and Li, Y. (2004) Structure-switching signaling aptamers: transducing molecular recognition into fluorescence signaling. Chem. Eur. J. 10:1868–1876.CrossRefGoogle Scholar
  59. 59.
    Nutiu, R. and Li, Y. (2005) In vitro selection of structure-switching signaling aptamers. Angew. Chem. Int. Ed. 44:1061–1065; S1061/1–S1061/3.CrossRefGoogle Scholar
  60. 60.
    Nutiu, R. and Li, Y. (2005) Aptamers with fluorescence-signaling properties. Methods 37:16–25.CrossRefGoogle Scholar
  61. 61.
    Liu, J. and Lu, Y. (2006) Smart nanomaterials responsive to multiple chemical stimuli with controllable cooperativity. Adv. Mater. 18:1667–1671.CrossRefGoogle Scholar
  62. 62.
    Levy, M., Cater, S.F. and Ellington, A.D. (2005) Quantum-dot aptamer beacons for the detection of proteins. ChemBioChem 6:2163–2166.CrossRefGoogle Scholar
  63. 63.
    Choi, J.H., Chen, K.H. and Strano, M.S. (2006) Aptamer-capped nanocrystal quantum dots: a new method for label-free protein detection. J. Am. Chem. Soc. 128:15584–15585.CrossRefGoogle Scholar
  64. 64.
    Dwarakanath, S., Bruno, J.G., Shastry, A., Phillips, T., John, A.A., Kumar, A. and Stephenson, L.D. (2004) Quantum dot-antibody and aptamer conjugates shift fluorescence upon binding bacteria. Biochem. Biophys. Res. Commun. 325:739–743.CrossRefGoogle Scholar
  65. 65.
    Shieh, F., Lavery, L., Chu, C.T., Richards-Kortum, R., Ellington, A.D. and Korgel, B.A. (2005) Semiconductor nanocrystal-aptamer bioconjugate probes for specific prostate carcinoma cell targeting. Proc. SPIE (Int. Soc. Opt. Eng.) 5705:159–165.Google Scholar
  66. 66.
    Chu, T.C., Shieh, F., Lavery, L.A., Levy, M., Richards-Kortum, R., Korgel, B.A. and Ellington, A.D. (2006) Labeling tumor cells with fluorescent nanocrystal-aptamer bioconjugates. Biosens. Bioelectron. 21:1859–1866.CrossRefGoogle Scholar
  67. 67.
    Mitchell, G.P., Mirkin, C.A. and Letsinger, R.L. (1999) Programmed assembly of DNA functionalized quantum dots. J. Am. Chem. Soc. 121:8122–8123.CrossRefGoogle Scholar
  68. 68.
    Gueroui, Z. and Libchaber, A. (2004) Single-molecule measurements of gold-quenched quantum dots. Phys. Rev. Lett. 93:166108/1–166108/4.CrossRefGoogle Scholar
  69. 69.
    Wargnier, R., Baranov, A.V., Maslov, V.G., Stsiapura, V., Artemyev, M., Pluot, M., Sukhanova, A. and Nabiev, I. (2004) Energy transfer in aqueous solutions of oppositely charged CdSe/ZnS core/shell quantum dots and in quantum dot-nanogold assemblies. Nano Lett. 4:451–457.CrossRefGoogle Scholar
  70. 70.
    Oh, E., Hong, M.-Y., Lee, D., Nam, S.-H., Yoon, H.C. and Kim, H.-S. (2005) Inhibition assay of biomolecules based on fluorescence resonance energy transfer (FRET) between quantum dots and gold nanoparticles. J. Am. Chem. Soc. 127:3270–3271.CrossRefGoogle Scholar
  71. 71.
    Dyadyusha, L., Yin, H., Jaiswal, S., Brown, T., Baumberg, J.J., Booy, F.P. and Melvin, T. (2005) Quenching of CdSe quantum dot emission, a new approach for biosensing. Chem. Commun. 25:3201–3203.CrossRefGoogle Scholar
  72. 72.
    Sato, K., Hosokawa, K. and Maeda, M. (2003) Rapid aggregation of gold nanoparticles induced by non-cross-linking DNA hybridization. J. Am. Chem. Soc. 125:8102–8103.CrossRefGoogle Scholar
  73. 73.
    Zhao, W., Chiuman, W., Brook, M.A. and Li, Y. (2007) Simple and rapid colorimetric biosensors based on DNA aptamer and noncrosslinking gold nanoparticle aggregation. ChemBioChem 8:727–731.CrossRefGoogle Scholar
  74. 74.
    Li, H. and Rothberg, L. (2004) Colorimetric detection of DNA sequences based on electrostatic interactions with unmodified gold nanoparticles. Proc. Natl. Acad. Sci. USA 101: 14036–14039.CrossRefGoogle Scholar
  75. 75.
    Li, H. and Rothberg, L.J. (2004) Label-free colorimetric detection of specific sequences in genomic DNA amplified by the polymerase chain reaction. J. Am. Chem. Soc. 126: 10958–10961.CrossRefGoogle Scholar
  76. 76.
    Li, H. and Rothberg, L.J. (2004) DNA sequence detection using selective fluorescence quenching of tagged oligonucleotide probes by gold nanoparticles. Anal. Chem. 76:5414–5417.CrossRefGoogle Scholar
  77. 77.
    Wang, L., Liu, X., Hu, X., Song, S. and Fan, C. (2006) Unmodified gold nanoparticles as a colorimetric probe for potassium DNA aptamers. Chem. Commun. 28:3780–3782.CrossRefGoogle Scholar
  78. 78.
    Glynou, K., Ioannou, P.C., Christopoulos, T.K. and Syriopoulou, V. (2003) Oligonucleotide-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for DNA analysis by hybridization. Anal. Chem. 75:4155–4160.CrossRefGoogle Scholar
  79. 79.
    Liu, J., Mazumdar, D. and Lu, Y. (2006) A simple and sensitive “dipstick” test in serum based on lateral flow separation of aptamer-linked nanostructures. Angew. Chem. Int. Ed. 45: 7955–7959.CrossRefGoogle Scholar
  80. 80.
    Famulok, M. and Mayer, G. (2006) Chemical biology: aptamers in nanoland. Nature (Lond.) 439:666–669.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Juewen Liu
  • Yi Lu

There are no affiliations available

Personalised recommendations