Biofunctional multimodal plasmonic nanostructures suitable for multiplexed localized surface plasmon resonance (LSPR) biosensing have been created by DNA-directed immobilization (DDI) of two distinct multifunctional biohybrid gold nanoparticles. Gold nanoparticles (AuNP) of distinct sizes, and therefore showing distinct plasmon resonant peaks (RP), have been biofunctionalized and codified with two different single stranded-DNA (ssDNA) chains. One of these oligonucleotide chains has been specifically designed to direct each AuNP to a distinct location of the surface of a DNA microarray chip through specific hybridization with complementary oligonucleotide strands. Scanning Electron Microscopy (SEM) has been used to demonstrate selective immobilization of each AuNP on distinct spots. The second ssDNA chain of the AuNPs provides the possibility to introduce by hybridization distinct types of bioactive molecules or bioreceptors, on a reversible manner. In this work, hapten-oligonucleotide bioconjugate probes, with sequences complementary to the second ssDNA linked to the AuNP, have been synthesized and used to create multiplexed hapten-biofuncionalized plasmonic nanostructures. The oligonucleotide probes consist on anabolic androgenic steroid haptens (AAS) covalently linked to specifically designed oligonucleotide sequences. The biofunctionality of these plasmonic nanostructures has been demonstrated by fluorescent microarray immunoassay and LSPR measurements, recording the shift of the RP produced after the antibody binding to the corresponding hapten-oligonucleotide probes immobilized on the nanostructured surface. Preliminary data show that this approach could allow manufacturing multifunctional multimodal LSPR chips for multiplexed analysis of different substances reaching very good detectability. Thus, small molecular weigh, analytes such as stanozolol (ST,) could be detected at concentrations in the low nM range. The results here presented open the door for an easy way to construct site-encoded multiplexed multimodal LSPR sensor transducers, combining the DDI strategies with multimodal biohybrid nanoparticles showing distinct optical properties.
Keywords: Anabolic-Androgenic Steroids (AAS); DNA-directed Immobilization (DDI); DNA-gold nanoparticles; Hapten-oligonucleotide bioconjugates; Localized surface plasmon resonance (LSPR); Multifunctional plasmonic nanostructures; Multiplexed biosensor.
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