title: Evaluation of direct versus multi-layer passivation and capture chemistries for nanoparticle-based biosensor applications.
abstract: Nanoparticles used in biosensor applications often fail when deployed directly in complex biological fluids. This is due to surface fouling and interference from the large concentration of non-specific binders (proteins, lipids, nucleic acids and saccharides) in the matrix. We systematically investigate four orthogonal approaches for decorating nanoparticle surfaces with affinity probes and evaluate their performance in buffer and serum. Carbodiimide coupling, cooper-mediated 'click' coupling, copper-free click coupling and thiol-maleimide coupling were quantitatively controlled during the fabrication process. Analyte mediated aggregation of fluorescent reporters and paramagnetic nanoparticle in a sandwich immunoassay was then used to probe assay sensitivity and specificity using an early biomarker of dengue fever, NS-1, as an exemplar and clinically relevant analyte. The type of surface functionalization played a vital role in assay performance in buffer versus serum at the assay sensitivity limit (3 ng/mL in serum) and over the linearity of response of the assay's dynamic range. There was a 10 fold increase on the dynamic range of the detection of NS1 comparing copper free click coupling to carbodiimide coupling, one of the most common approaches for nanoparticle functionalization. By tuning their size, we could carefully monitor the evolution of nanoparticle populations by flow cytometer and discriminate between unbound and fluorescent nanoparticles. This subtle control on each assay component resulted in more than a 10-fold reduction in fluorescence background and improved the sensitivity of almost two orders of magnitude compared to endpoint measurements.
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, Brisbane, 4072 QLD, Australia.
authors.forename: K C
authors.initials: KC
authors.lastname: Sanjaya
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, Brisbane, 4072 QLD, Australia.
authors.forename: Andrea
authors.initials: A
authors.lastname: Ranzoni
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, Brisbane, 4072 QLD, Australia; School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, 4072 QLD, Australia.
authors.forename: Daniel
authors.initials: D
authors.lastname: Watterson
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, Brisbane, 4072 QLD, Australia; School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, 4072 QLD, Australia.
authors.forename: Paul
authors.initials: P
authors.lastname: Young
authors.affiliation: Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, Brisbane, 4072 QLD, Australia. Electronic address: m.cooper@uq.edu.au.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Cooper
mesh_terms: D015374:Biosensing Techniques; D002241:Carbohydrates / Q000737:chemistry; D057930:Click Chemistry; D005453:Fluorescence; D006801:Humans; D007118:Immunoassay; D008055:Lipids / Q000737:chemistry; D053758:Nanoparticles / Q000737:chemistry*; D009696:Nucleic Acids / Q000737:chemistry; D011506:Proteins / Q000737:chemistry; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D002241:Carbohydrates; D008055:Lipids; D009696:Nucleic Acids; D011506:Proteins; D013438:Sulfhydryl Compounds
keywords: Biosensor; Coupling chemistry; Dengue; Immunoassay; Nanoparticle; Paramagnetic; Surface chemistry