1. Double-Resonant Nanostructured Gold Surface for Multiplexed Detection.
- Author
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Minopoli A, Scardapane E, Ventura BD, Tanner JA, Offenhäusser A, Mayer D, and Velotta R
- Subjects
- Antibodies, Immobilized chemistry, Antibodies, Immobilized immunology, Aptamers, Nucleotide chemistry, Aptamers, Nucleotide metabolism, Carbocyanines chemistry, Fluoresceins chemistry, Glass chemistry, Humans, Immunoassay methods, L-Lactate Dehydrogenase blood, L-Lactate Dehydrogenase immunology, Limit of Detection, Plasmodium falciparum enzymology, Protozoan Proteins blood, Protozoan Proteins immunology, Surface Properties, Gold chemistry, Metal Nanoparticles chemistry
- Abstract
A novel double-resonant plasmonic substrate for fluorescence amplification in a chip-based apta-immunoassay is herein reported. The amplification mechanism relies on plasmon-enhanced fluorescence (PEF) effect. The substrate consists of an assembly of plasmon-coupled and plasmon-uncoupled gold nanoparticles (AuNPs) immobilized onto a glass slide. Plasmon-coupled AuNPs are hexagonally arranged along branch patterns whose resonance lies in the red band (∼675 nm). Plasmon-uncoupled AuNPs are sprinkled onto the substrate, and they exhibit a narrow resonance at 524 nm. Numerical simulations of the plasmonic response of the substrate through the finite-difference time-domain (FDTD) method reveal the presence of electromagnetic hot spots mainly confined in the interparticle junctions. In order to realize a PEF-based device for potential multiplexing applications, the plasmon resonances are coupled with the emission peak of 5-carboxyfluorescein (5-FAM) fluorophore and with the excitation/emission peaks of cyanine 5 (Cy5). The substrate is implemented in a malaria apta-immunoassay to detect Plasmodium falciparum lactate dehydrogenase ( Pf LDH) in human whole blood. Antibodies against Plasmodium biomarkers constitute the capture layer, whereas fluorescently labeled aptamers recognizing Pf LDH are adopted as the top layer. The fluorescence emitted by 5-FAM and Cy5 fluorophores are linearly correlated (logarithm scale) to the Pf LDH concentration over five decades. The limits of detection are 50 pM (1.6 ng/mL) with the 5-FAM probe and 260 fM (8.6 pg./mL) with the Cy5 probe. No sample preconcentration and complex pretreatments are required. Average fluorescence amplifications of 160 and 4500 are measured in the 5-FAM and Cy5 channel, respectively. These results are reasonably consistent with those worked out by FDTD simulations. The implementation of the proposed approach in multiwell-plate-based bioassays would lead to either signal redundancy (two dyes for a single analyte) or to a simultaneous detection of two analytes by different dyes, the latter being a key step toward high-throughput analysis.
- Published
- 2022
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