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Micro and nanotechnology for early diagnosis and detection of rheumatic diseases-molecular markers


Cytokine proteins are known as biomarker molecules, characteristic of a disease or specific body condition. Monitoring of the cytokine pattern in body fluids contributes to the diagnosis of rheumatic diseases associated with rheumatoid factor. Here, the proinflammatory cytokine Tumor Necrosis Factor alpha (TNF-α) was chosen as the first target of interest. We report on the development of a fast sandwich immunosorbent assay: anti-TNF-α antibody (Ab) immobilization on a pre-activated 3D micro-structured glass slide, coupled with direct fluorescence readout method. Two differently functionalized glass - Ab-immobilization substrates - were compared: O2 PLASMA/APTES activated glass - having a two-dimensional structure-, and the polystyrene (PS) beads coated glass, -having a 3D structure-. The 3D structured substrate was aimed to increase antibody anchorage sites. Then, the optimization of buffer media was carefully investigated with respect to non-specific protein binding. As a first step towards real sample analysis, a proof of principle of on/off cytokine detection has been analyzed in the presence of human serum. High-density one-step immobilization of anti-TNF-α antibody onto nanobead-coated glass slide chip has been demonstrated to be a promising device for application in in vitro diagnostic and profiling of cytokines with potential application toward personalized therapeutic interventions.

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  1. 1.

    Brennan, F.M. & McInnes, I.B. Evidence that cytokines plays a role in rheumatoid arthritis. J. Clin. Invest. 118, 3537–3545 (2008).

    CAS  Article  Google Scholar 

  2. 2.

    Firestein, G.S. Evolving concepts of rheumatoid arthritis. Nature 423, 356–361 (2003).

    CAS  Article  Google Scholar 

  3. 3.

    Ramanjaneya, V.R.M. & Shashidharamurthy, R. Multifaced roles of TNF-α during the pathogenesis of rheumatoid arthritis. Adv. Biosci. Biotechnol. 4, 937–940 (2013).

    Article  Google Scholar 

  4. 4.

    Vasanthi, P., Nalini, G. & Rajasekhar, G. Role of tumor necrosis factor-alpha in rheumatoid arthritis: a review. APLAR Journal of Rheumatology 10, 270–274 (2007).

    Article  Google Scholar 

  5. 5.

    Matsuno, H. et al. The role of TNF-alpha in the pathogenesis of inflammation and joint destruction in rheumatoid arthritis (RA): a study using a human RA/SCID mouse chimera. Rheumatology (Oxford) 41, 329–337 (2002).

    CAS  Article  Google Scholar 

  6. 6.

    Moelants, E.A.V., Mortier, A., Van Damme, J. & Proost, P. Regulation of TNF-α with a focus on rheumatoid arthritis. Immunol. Cell Biol. 91, 393–401 (2013).

    CAS  Article  Google Scholar 

  7. 7.

    Lindstrom, T.M. & Robinson, W.H. Biomarkers for rheumatoid arthritis: making it personal. Scand. J. Clin. Lab. Invest. Suppl. 242, 79–84 (2010).

    Article  Google Scholar 

  8. 8.

    Edrees, A.F., Misra, S.N. & Abdou, N.I. Anti-tumor necrosis factor (TNF) therapy in rheumatoid arthritis: correlation of TNF-alpha serum level with clinical response and benefit from changing dose or frequency of infliximab infusions. Clin. Exp. Rheumatol. 23, 469–474 (2005).

    CAS  Google Scholar 

  9. 9.

    Leng, S.X. et al. Elisa and multiplex technologies for cytokine measurement in inflammation and aging research. J. Gerontol. A Biol. Sci. Med. Sci. 63, 879–884 (2008).

    Article  Google Scholar 

  10. 10.

    Rusmini, F., Zhong, Z. & Feijen, J. Protein immobilization strategies for protein biochips. Biomacromolecules 8, 1775–1789 (2007).

    CAS  Article  Google Scholar 

  11. 11.

    Kim, D. & Herr, A.E. Protein immobilization techniques for microfluidic assays. Biomicrofluidics 7, 41501 (2013).

    Article  Google Scholar 

  12. 12.

    Liu, Y. & Li, C.M. Advanced immobilization and amplification for high performance protein chips. Analytical Letters 45, 130–155 (2012).

    Article  Google Scholar 

  13. 13.

    Sung, D., Yang, S., Park, J. & Jon, S. High-density immobilization of antibodies onto nanobead-coated cyclic olefin copolymer plastic surfaces for application as a sensitive immunoassay chip. Biomed. Microdevices 15, 691–698 (2013).

    CAS  Article  Google Scholar 

  14. 14.

    Xu, B., Shuai, C., Xianhua, W., Dongmei, W. & Liang, X. Novel polystyrene/antibody nanoparticle-coated capillary for immunoaffinity in-tube solid-phase microextraction. Anal. Bioanal. Chem. 407, 2771–2775 (2015).

    CAS  Article  Google Scholar 

  15. 15.

    Vashist, S.K., Schneider, E.M., Lam, E., Hrapovic, S. & Luong, J.H.T. One-step antibody immobilizationbased rapid and highly-sensitive sandwich ELISA procedure for potential in vitro diagnostics. Sci. Rep. 4, 4407 (2014).

    Article  Google Scholar 

  16. 16.

    Dixit, C.K., Saraswat, M. & Holthofer, H. Evaluation of apparent non-specific protein loss due to adsorption on sample tube surfaces and/or altered immunogenicity. Analyst 136, 1406–1411 (2011).

    CAS  Article  Google Scholar 

  17. 17.

    Arkles, B., Steinmetz, J.R., Zazyczny, J. & Mehta, P. Factors contributing to the stability of alkoxysilanes in aqueous solution. J. Adhes. Sci. Technol. 6, 193–206 (1992).

    CAS  Article  Google Scholar 

  18. 18.

    Derjaguin, B.V., Muller, V.M. & Toporov, Y.P. Effect of contact deformations on the adhesion of particles. J. Colloid Interface Sci. 53, 314–326 (1975).

    CAS  Article  Google Scholar 

  19. 19.

    Ebrahimi, A.A. et al. Serum levels of TNF-alpha, TNF-alphaRI, TNF-alphaRII and IL-12 in treated rheumatoid arthritis patients. Iran. J. Immunol. 6, 147–153 (2009).

    CAS  Google Scholar 

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Correspondence to Rosaria Rinaldi.

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These authors contributed equally to this work.

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Marulli, E., Aloisi, A., Di Giuseppe, P. et al. Micro and nanotechnology for early diagnosis and detection of rheumatic diseases-molecular markers. BioChip J 10, 189–197 (2016).

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  • Immunoassay
  • Polystyrene beads
  • Chip
  • TNF-α
  • Rheumatic diseases