Analytical and Bioanalytical Chemistry

, Volume 410, Issue 4, pp 1397–1403 | Cite as

Ultrasensitive colorimetric detection of NF-κB protein at picomolar levels using target-induced passivation of nanoparticles

  • P. Abdul Rasheed
  • Jae-Seung LeeEmail author
Research Paper


We developed a highly sensitive and selective sensor based on the nanoprobe conjugates of catalytic nanoparticles and double-stranded DNA (dsDNA) for the colorimetric detection of NF-κB protein. The sensing mechanism takes advantage of the catalytic activity of nanoparticle surfaces and the specific binding of NF-κB to a dsDNA sequence. In the presence of NF-κB, the highly selective interactions between dsDNA and NF-κB lead to the passivation of the catalytic nanoparticle surfaces, impeding the sodium borohydride-mediated reduction rate of 4-nitrophenol. The correlation between the NF-κB concentration and the visualized reduction rate of 4-nitrophenol from yellow to colorless clearly demonstrates the highly quantitative nature of the sensor. Importantly, this sensor can conclusively detect concentrations as low as 6.39 pM of NF-κB, which to best of our knowledge is the lowest limit of detection for a colorimetric NF-κB detection system. The excellent sensitivity of this sensor relies on the high binding constant of NF-κB to dsDNA and the catalytic activity of nanoparticle surfaces for the signal amplification. This sensor allows visual detection without the need for any spectrometric instrumentation. We also determined the various parameters such as the pH, temperature, incubation time, and salt concentration for optimal NF-κB-dsDNA interactions. Finally, we demonstrated the performance of the sensor with simulated sample analysis.

Graphical abstract

A highly sensitive and selective colorimetric detection of protein NF-κB using the nanoprobeconjugates of catalytic gold nanoparticles and double-stranded DNA (dsDNA) has been developed.


Gold nanoparticle DNA Colorimetric detection Protein 



This work was supported by the NRF funded by the Korean government, MSIP (NRF-2015R1C1A1A01053865, NRF-2015M3A9D7031015, and NRF-2016R1A5A1010148).

Compliance with ethical standards

Conflicts of Interest

The authors declare no conflict of interest.

Supplementary material

216_2017_783_MOESM1_ESM.pdf (324 kb)
ESM 1 (PDF 323 kb)


  1. 1.
    Pan Q, Zhang RY, Bai YF, He NY, Lu ZH. An electrochemical approach for detection of specific DNA-binding protein by gold nanoparticle-catalyzed silver enhancement. Anal Biochem. 2008;375(2):179–86.CrossRefGoogle Scholar
  2. 2.
    Vallee-Belisle A, Bonham AJ, Reich NO, Ricci F, Plaxco KW. Transcription factor beacons for the quantitative detection of DNA binding activity. J Am Chem Soc. 2011;133(35):13836–9.CrossRefGoogle Scholar
  3. 3.
    Giannetti A, Citti L, Domenici C, Tedeschi L, Baldini F, Wabuyele MB, et al. FRET-based protein-DNA binding assay for detection of active NF-kappa B. Sensors Actuators B Chem. 2006;113(2):649–54.CrossRefGoogle Scholar
  4. 4.
    Ma DL, Xu T, Chan DSH, Man BYW, Fong WF, Leung CH. A highly selective, label-free, homogenous luminescent switch-on probe for the detection of nanomolar transcription factor NF-kappaB. Nucleic Acids Res. 2011;39(10):e67.CrossRefGoogle Scholar
  5. 5.
    Pineda-Molina E, Klatt P, Vazquez J, Marina A, de Lacoba MG, Perez-Sala D, et al. Glutathionylation of the p50 subunit of NF-kappa B: a mechanism for redox-induced inhibition of DNA binding. Biochemistry. 2001;40(47):14134–42.CrossRefGoogle Scholar
  6. 6.
    Badr CE, Niers JM, Tjon-Kon-Fat LA, Noske DP, Wurdinger T, Tannous BA. Real-Time monitoring of nuclear factor kappa b activity in cultured cells and in animal models. Mol Imaging. 2009;8(5):278–90.Google Scholar
  7. 7.
    Kumar A, Takada Y, Boriek AM, Aggarwal BB. Nuclear factor-kappa B: its role in health and disease. J Mol Med. 2004;82(7):434–48.CrossRefGoogle Scholar
  8. 8.
    Bharti AC, Aggarwal BB. Nuclear factor-kappa B and cancer: its role in prevention and therapy. Biochem Pharmacol. 2002;64(5/-6):883–8.CrossRefGoogle Scholar
  9. 9.
    Huang Y, Chen J, Zhao SL, Shi M, Chen ZF, Liang H. Label-free colorimetric aptasensor based on nicking enzyme assisted signal amplification and DNAzyme amplification for highly sensitive detection of protein. Anal Chem. 2013;85(9):4423–30.CrossRefGoogle Scholar
  10. 10.
    Gao T, Wang L, Zhu J, Zhu DS, Jiang W. Fok I cleavage-inhibition strategy for the specific and accurate detection of transcription factors. Talanta. 2015;144:44–50.CrossRefGoogle Scholar
  11. 11.
    Hellman LM, Fried MG. Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions. Nat Protoc. 2007;2(8):1849–61.CrossRefGoogle Scholar
  12. 12.
    Hampshire AJ, Rusling DA, Broughton-Head VJ, Fox KR. Footprinting: a method for determining the sequence selectivity, affinity and kinetics of DNA-binding ligands. Methods. 2007;42(2):128–40.CrossRefGoogle Scholar
  13. 13.
    Fried M, Crothers DM. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide-gel electrophoresis. Nucleic Acids Res. 1981;9(23):6505–25.CrossRefGoogle Scholar
  14. 14.
    Gupta SV, McGowen RM, Callewaert DM, Brown TR, Li YW, Sarkar FH. Quantitative chemiluminescent immunoassay for NF-kappa B-DNA binding activity. J Immunoass Immunochem. 2005;26(2):125–43.CrossRefGoogle Scholar
  15. 15.
    Metelev V, Zhang SR, Tabatadze D, Bogdanov A. Hairpin-like fluorescent probe for imaging of NF-kappa B transcription factor activity. Bioconjug Chem. 2011;22(4):759–65.CrossRefGoogle Scholar
  16. 16.
    Liu XF, Lan OY, Cai XH, Huang YQ, Feng XM, Fan QL, et al. An ultrasensitive label-free biosensor for assaying of sequence-specific DNA-binding protein based on amplifying fluorescent conjugated polymer. Biosens Bioelectron. 2013;41:218–24.CrossRefGoogle Scholar
  17. 17.
    Chen JH, Zhang X, Cai SX, Wu DZ, Lin J, Li CY, et al. Label-free electrochemical biosensor using home-made 10-methyl-3-nitro-acridone as indicator for picomolar detection of nuclear factor kappa B. Biosens Bioelectron. 2014;53:12–7.CrossRefGoogle Scholar
  18. 18.
    Li J, Fu HE, Wu LJ, Zheng AX, Chen GN, Yang HH. General colorimetric detection of proteins and small molecules based on cyclic enzymatic signal amplification and hairpin aptamer probe. Anal Chem. 2012;84(12):5309–15.CrossRefGoogle Scholar
  19. 19.
    Vilela D, Gonzalez MC, Escarpa A. Sensing colorimetric approaches based on gold and silver nanoparticles aggregation: chemical creativity behind the assay. A review. Anal Chim Acta. 2012;751:24–43.CrossRefGoogle Scholar
  20. 20.
    Polavarapu L, Perez-Juste J, Xu QH, Liz-Marzan LM. Optical sensing of biological, chemical and ionic species through aggregation of plasmonic nanoparticles. J Mater Chem C. 2014;2(36):7460–76.CrossRefGoogle Scholar
  21. 21.
    Zhang HQ, Li F, Dever B, Li XF, Le XC. DNA-mediated homogeneous binding assays for nucleic acids and proteins. Chem Rev. 2013;113(4):2812–41.CrossRefGoogle Scholar
  22. 22.
    Chang CC, Chen CY, Chuang TL, Wu TH, Wei SC, Liao H, et al. Aptamer-based colorimetric detection of proteins using a branched DNA cascade amplification strategy and unmodified gold nanoparticles. Biosens Bioelectron. 2016;78:200–5.CrossRefGoogle Scholar
  23. 23.
    Rasheed PA, Sandhyarani N. Femtomolar level detection of BRCA1 gene using a gold nanoparticle labeled sandwich type DNA sensor. Colloids Surf B: Biointerfaces. 2014;117:7–13.CrossRefGoogle Scholar
  24. 24.
    Rasheed PA, Sandhyarani N. Attomolar detection of BRCA1 gene based on gold nanoparticle assisted signal amplification. Biosens Bioelectron. 2015;65:333–40.CrossRefGoogle Scholar
  25. 25.
    Ahn J, Choi Y, Lee AR, Lee JH, Jung JH. A duplex DNA-gold nanoparticle probe composed as a colorimetric biosensor for sequence-specific DNA-binding proteins. Analyst. 2016;141(6):2040–5.CrossRefGoogle Scholar
  26. 26.
    Degliangeli F, Kshirsagar P, Brunetti V, Pompa PP, Fiammengo R. Absolute and direct microRNA quantification using DNA-gold nanoparticle probes. J Am Chem Soc. 2014;136(6):2264–7.CrossRefGoogle Scholar
  27. 27.
    Yang XF, Li J, Pei H, Zhao Y, Zuo XL, Fan CH, et al. DNA-gold nanoparticle conjugates-based nanoplasmonic probe for specific differentiation of cell types. Anal Chem. 2014;86(6):3227–31.CrossRefGoogle Scholar
  28. 28.
    Zhang Y, Hu J, Zhang CY. Sensitive detection of transcription factors by isothermal exponential amplification-based colorimetric assay. Anal Chem. 2012;84(21):9544–9.CrossRefGoogle Scholar
  29. 29.
    Cuenya BR. Metal nanoparticle catalysts beginning to shape-up. Acc Chem Res. 2013;46(8):1682–91.CrossRefGoogle Scholar
  30. 30.
    Kim BH, Yoon IS, Lee JS. Masking nanoparticle surfaces for sensitive and selective colorimetric detection of proteins. Anal Chem. 2013;85(21):10542–8.CrossRefGoogle Scholar
  31. 31.
    Oh JH, Lee JS. Designed hybridization properties of DNA-gold nanoparticle conjugates for the ultraselective detection of a single-base mutation in the breast cancer gene BRCA1. Anal Chem. 2011;83(19):7364–70.CrossRefGoogle Scholar
  32. 32.
    Kim JY, Lee JS. Synthesis and thermally reversible assembly of DNA-gold nanoparticle cluster conjugates. Nano Lett. 2009;9(12):4564–9.CrossRefGoogle Scholar
  33. 33.
    Siggers T, Chang AB, Teixeira A, Wong D, Williams KJ, Ahmed B, et al. Principles of dimer-specific gene regulation revealed by a comprehensive characterization of NF-kappa B family DNA binding. Nat Immunol. 2012;13(1):95–U123.CrossRefGoogle Scholar
  34. 34.
    Siggers T, Gordan R. Protein-DNA binding: complexities and multi-protein codes. Nucleic Acids Res. 2014;42(4):2099–111.CrossRefGoogle Scholar
  35. 35.
    Gilmore TD. Introduction to NF-kappa B: players, pathways, perspectives. Oncogene. 2006;25(51):6680–4.CrossRefGoogle Scholar
  36. 36.
    Zabel U, Schreck R, Baeuerle PA. DNA-binding of purified transcription factor NF-kB – affinity, specificity, Zn2+ dependence, and differential half-site recognition. J Biol Chem. 1991;266(1):252–60.Google Scholar
  37. 37.
    Phelps CB, Sengchanthalangsy LL, Malek S, Ghosh G. Mechanism of kappa B DNA binding by Rel/NF-kappa B dimers. J Biol Chem. 2000;275(32):24392–9.CrossRefGoogle Scholar
  38. 38.
    Li J, Liu CY, Liu Y. Au/graphene hydrogel: synthesis, characterization and its use for catalytic reduction of 4-nitrophenol. J Mater Chem. 2012;22(17):8426–30.CrossRefGoogle Scholar
  39. 39.
    Freshney RI. Culture of animal cells: a manual of basic technique. 3rd ed. New York: Wiley-Liss; 1994.Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Department of Materials Science and EngineeringKorea UniversitySeoulRepublic of Korea

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