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Sensing of proteins in human serum using conjugates of nanoparticles and green fluorescent protein

Abstract

There is a direct correlation between protein levels and disease states in human serum, which makes it an attractive target for sensors and diagnostics. However, this is challenging because serum features more than 20,000 proteins, with an overall protein content greater than 1 mM. Here we report a sensor based on a hybrid synthetic–biomolecule that uses arrays of green fluorescent protein and nanoparticles to detect proteins at biorelevant concentrations in both buffer and human serum. Distinct and reproducible fluorescence-response patterns were obtained from five serum proteins (human serum albumin, immunoglobulin G, transferrin, fibrinogen and α-antitrypsin), both in buffer and when spiked into human serum. Using linear discriminant analysis we identified these proteins with an identification accuracy of 100% in buffer and 97% in human serum. The arrays were also able to discriminate between different concentrations of the same protein, as well as a mixture of different proteins in human serum.

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Figure 1: Modes of sensor response and structural features of NPs.
Figure 2: Array-based sensing of five serum proteins in 5 mM sodium phosphate buffer (pH 7.40).
Figure 3: Determination of the optimum ratio of fluorophore to NP concentration.
Figure 4: Array-based sensing of five serum proteins in human serum.
Figure 5: Discrimination of HSA and IgG at different concentrations and mixtures of proteins.

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References

  1. Hardy, J. & Selkoe, D. J. Medicine – the amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297, 353–356 (2002).

    Article  CAS  PubMed  Google Scholar 

  2. Pulido, R. & van Huijsduijnen, R. H. Protein tyrosine phosphatases: dual-specificity phosphatases in health and disease. FEBS J. 275, 848–866 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Adkins, J. N. et al. Toward a human blood serum proteome – analysis by multidimensional separation coupled with mass spectrometry. Mol. Cell. Proteomics 1, 947–955 (2002).

    Article  CAS  PubMed  Google Scholar 

  4. Pieper, R. et al. The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins. Proteomics 3, 1345–1364 (2003).

    Article  CAS  PubMed  Google Scholar 

  5. Antman, E. M. et al. Cardiac-specific troponin I levels to predict the risk of mortality in patients with acute coronary syndromes. N. Engl. J. Med. 335, 1342–1349 (1996).

    Article  CAS  PubMed  Google Scholar 

  6. Anderson, G. P. et al. Quantifying serum antiplague antibody with a fiber-optic biosensor. Clin. Diagn. Lab. Immunol. 5, 609–612 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Badgwell, D. & Bast, R. C. Early detection of ovarian cancer. Dis. Markers 23, 397–410 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Murphy, G. P., Elgamal, A. A. A., Su, S. L., Bostwick, D. G. & Holmes, E. H. Current evaluation of the tissue localization and diagnostic utility of prostate specific membrane antigen. Cancer 83, 2259–2269 (1998).

    Article  CAS  PubMed  Google Scholar 

  9. McPherson, R. A. & Pincus, M. R. Henry's Clinical Diagnosis and Management by Laboratory Methods Ch 19 (Saunders–Elsevier, 2007).

    Google Scholar 

  10. Li, J. N., Zhang, Z., Rosenzweig, J., Wang, Y. Y. & Chan, D. W. Proteomics and bioinformatics approaches for identification of serum biomarkers to detect breast cancer. Clin. Chem. 48, 1296–1304 (2002).

    CAS  PubMed  Google Scholar 

  11. Baggerly, K. A., Morris, J. S. & Coombes, K. R. Reproducibility of SELDI–TOF protein patterns in serum: comparing datasets from different experiments. Bioinformatics 20, 777–U710 (2004).

    Article  CAS  PubMed  Google Scholar 

  12. Wright, A. T., Zhong, Z. & Anslyn, E. V. A functional assay for heparin in serum using a designed synthetic receptor. Angew. Chem. Int. Ed. 44, 5679–5682 (2005).

    Article  CAS  Google Scholar 

  13. Tobey, S. L. & Anslyn, E. V. Determination of inorganic phosphate in serum and saliva using a synthetic receptor. Org. Lett. 5, 2029–2031 (2003).

    Article  CAS  PubMed  Google Scholar 

  14. Wright, A. T. & Anslyn, E. V. Differential receptor arrays and assays for solution-based molecular recognition. Chem. Soc. Rev. 35, 14–28 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Lavigne, J. J. & Anslyn, E. V. Sensing a paradigm shift in the field of molecular recognition: from selective to differential receptors. Angew. Chem. Int. Ed. 40, 3119–3130 (2001).

    Article  Google Scholar 

  16. Wright, A. T., Edwards, N. Y., Anslyn, E. V. & McDevitt, J. T. The discriminatory power of differential receptor arrays is improved by prescreening – a demonstration in the analysis of tachykinins and similar peptides. Angew. Chem. Int. Ed. 46, 8212–8215 (2007).

    Article  CAS  Google Scholar 

  17. Zhou, H. C., Baldini, L., Hong, J., Wilson, A. J. & Hamilton, A. D. Pattern recognition of proteins based on an array of functionalized porphyrins. J. Am. Chem. Soc. 128, 2421–2425 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Miranda, O. R. et al. Array-based sensing of proteins using conjugated polymers. J. Am. Chem. Soc. 129, 9856–9857 (2007).

    Article  CAS  PubMed  Google Scholar 

  19. Stephenson, C. J. & Shimizu, K. D. Colorimetric and fluorometric molecularly imprinted polymer sensors and binding assays. Polym. Int. 56, 482–488 (2007).

    Article  CAS  Google Scholar 

  20. You, C. C. et al. Detection and identification of proteins using nanoparticle–fluorescent polymer ‘chemical nose’ sensors. Nature Nanotechnol. 2, 318–323 (2007).

    Article  CAS  Google Scholar 

  21. Phillips, R. L., Miranda, O. R., You, C. C., Rotello, V. M. & Bunz, U. H. F. Rapid and efficient identification of bacteria using gold-nanoparticle–poly(para-phenyleneethynylene) constructs. Angew. Chem. Int. Ed. 47, 2590–2594 (2008).

    Article  CAS  Google Scholar 

  22. De, M., You, C. C., Srivastava, S. & Rotello, V. M. Biomimetic interactions of proteins with functionalized nanoparticles: a thermodynamic study. J. Am. Chem. Soc. 129, 10747–10753 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Hong, R. et al. Control of protein structure and function through surface recognition by tailored nanoparticle scaffolds. J. Am. Chem. Soc. 126, 739–743 (2004).

    Article  CAS  PubMed  Google Scholar 

  24. Tsien, R. Y. The green fluorescent protein. Annu. Rev. Biochem. 67, 509–544 (1998).

    Article  CAS  PubMed  Google Scholar 

  25. Chalfie, M. & Kain, S. R. Green Fluorescent Protein: Properties, Applications, and Protocols p. 69 (Wiley-Interscience, 2006).

    Google Scholar 

  26. Anderson, N. L. & Anderson, N. G. The human plasma proteome – history, character, and diagnostic prospects. Mol. Cell. Proteomics 1, 845–867 (2002).

    Article  CAS  PubMed  Google Scholar 

  27. You, C. C., De, M., Han, G. & Rotello, V. M. Tunable inhibition and denaturation of alpha-chymotrypsin with amino acid-functionalized gold nanoparticles. J. Am. Chem. Soc. 127, 12873–12881 (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Jurs, P. C., Bakken, G. A. & McClelland, H. E. Computational methods for the analysis of chemical sensor array data from volatile analytes. Chem. Rev. 100, 2649–2678 (2000).

    Article  CAS  PubMed  Google Scholar 

  29. De, M., Rana, S. & Rotello, V. M. Nickel-ion-mediated control of the stoichiometry of his-tagged protein/nanoparticle interactions. Macromol. Biosci. 9, 174–178 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. SYSTAT 11.0 (Systat Software, Richmond, CA 94804, USA, 2004).

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Acknowledgements

This work was supported by the National Science Foundation Center for Hierarchical Manufacturing at the University of Massachusetts and the National Institutes of Health.

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Contributions

M.D., S.R. and V.R. conceived and designed the experiments. M.D., S.R., H.A., O.M. and R.A. performed the experiments. M.D., S.R., O.M. and V.R. analysed the data. M.D., S.R., U.B. and V.R. co-wrote the paper.

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Correspondence to Vincent M. Rotello.

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De, M., Rana, S., Akpinar, H. et al. Sensing of proteins in human serum using conjugates of nanoparticles and green fluorescent protein. Nature Chem 1, 461–465 (2009). https://doi.org/10.1038/nchem.334

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