Multifunctional nanoparticles as simulants for a gravimetric immunoassay |
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Authors: | Scott A Miller Leslie A Hiatt Robert G Keil David W Wright David E Cliffel |
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Institution: | (1) Department of Chemistry, Vanderbilt University, 7330 Stevenson Center, VU Station B 351822, Nashville, TN 37235-1822, USA;(2) Present address: Department of Chemistry, University of South Alabama, Mobile, AL 36688, USA;(3) Present address: Department of Chemistry, University of Dayton, Dayton, OH 45469, USA; |
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Abstract: | Immunoassays are important tools for the rapid detection and identification of pathogens, both clinically and in the research
laboratory. An immunoassay with the potential for the detection of influenza was developed and tested using hemagglutinin
(HA), a commonly studied glycoprotein found on the surface of influenza virions. Gold nanoparticles were synthesized, which
present multiple peptide epitopes, including the HA epitope, in order to increase the gravimetric response achieved with the
use of a QCM immunosensor for influenza. Specifically, epitopes associated with HA and FLAG peptides were affixed to gold
nanoparticles by a six-mer PEG spacer between the epitope and the terminal cysteine. The PEG spacer was shown to enhance the
probability for interaction with antibodies by increasing the distance the epitope extends from the gold surface. These nanoparticles
were characterized using thermogravimetric analysis, transmission electron microscopy, matrix-assisted laser desorption/ionization-time
of flight, and 1H nuclear magnetic resonance analysis. Anti-FLAG and anti-HA antibodies were adhered to the surface of a QCM, and the response
of each antibody upon exposure to HA, FLAG, and dual functionalized nanoparticles was compared with binding of Au–tiopronin
nanoparticles and H5 HA proteins from influenza virus (H5N1). Results demonstrate that the immunoassay was capable of differentiating
between nanoparticles presenting orthogonal epitopes in real-time with minimal nonspecific binding. The detection of H5 HA
protein demonstrates the logical extension of using these nanoparticle mimics as a safe positive control in the detection
of influenza, making this a vital step in improving influenza detection methodology. |
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