Gold Nanostars Bioconjugation for Selective Targeting and SERS Detection of Biofluids

Abstract

Gold nanoparticles (AuNPs) show physicochemical and optical functionalities that are of great interest for spectroscopy-based detection techniques, and especially for surface enhanced Raman spectroscopy (SERS), which is capable of providing detailed information on the molecular content of analysed samples. Moreover, the introduction of different moieties combines the interesting plasmonic properties of the AuNPs with the specific and selective recognition capabilities of the antibodies (Ab) towards antigens. The conjugation of biomolecules to gold nanoparticles (AuNPs) has received considerable attention for analysis of liquid samples and in particular biological fluids (biofluids) in clinical diagnostic and therapeutic field. To date, gold nanostars (AuNSts) are gaining more and more attention as optimal enhancers for SERS signals due to the presence of sharp branches protruding from the core, providing a huge number of “hot spots”. To this end, we focused our attention on the design, optimization, and deep characterization of a bottom up-process for (i) AuNPs increasing stabilization in high ionic strength buffer, (ii) covalent conjugation with antibodies, while (iii) retaining the biofunctionality to specific tag analyte within the biofluids. In this work, a SERS-based substrate was developed for the recognition of a short fragment (HA) of the hemagglutinin protein, which is the major viral antigen inducing a neutralizing antibody response. The activity and specific targeting with high selectivity of the Ab-AuNPs was successfully tested in transfected neuroblastoma cells cultures. Then, SERS capabilities were assessed measuring Raman spectra of HA solution, thus opening interesting perspective for the development of novel versatile highly sensitive biofluids sensors.

Design and Synthesis of Novel Raman Reporters for Bioorthogonal SERS Nanoprobes Engineering

“…To this end, while PEG/NP ratio was kept constant at 500 k to achieve the 100% particle coverage as previously demonstrated [ 6 ], different RR/NP ratios were investigated to study the effect of RRs at 5 k, 10 k, 25 k, and 50 k ratios. Optimal PEG/NPs/RRs incubation conditions were those giving brighter bioorthogonal Raman signals while retaining SERS nanotags morphology and avoiding particle aggregation.…”

“…Taking advantages of surface-localized plasmons phenomena occurring upon metallic nanoparticles (NPs)–light interactions, the Raman signal scattered from target analytes that are adsorbed onto SERS NPs is enhanced (up to 10 6 ) with respect to the overall signal from the background biomatrices. Consequently, in order to improve the sensitivity and specificity of the technique, the surface chemistry of NPs can be engineered to increase the affinity toward the biomarkers of interest [ 6 ]. Among all reported morphologies, highly branched star-shaped gold particles (AuNSts) are particularly appealing SERS substrates due to a large number of intrinsic hotspots and the high surface-to-volume ratio compared to isotropic spherical NPs [ 7 ].…”

“…Among all reported morphologies, highly branched star-shaped gold particles (AuNSts) are particularly appealing SERS substrates due to a large number of intrinsic hotspots and the high surface-to-volume ratio compared to isotropic spherical NPs [ 7 ]. Furthermore, Au surfaces provide an interface for easy covalent conjugation of biomolecules (e.g., antibodies) by thiol-gold chemistry [ 6 , 8 ]. Consolidated gold functionalization strategies can be exploited to further decorate NPs surfaces with Raman reporters (RRs), a special class of compounds whose spectral signature exhibit very narrow bandwidth (~1 ÷ 2 nm) and intense peaks [ 9 , 10 ].…”

“…RRs NPs loading was optimized to guarantee the highest signal from alkyne- and cyano-derived moieties of the aromatic substituents acting as strong NIR emitting Raman signals with a limit of detection at concentration down to pM. SERS nanotags can provide selective biological tags by simply assembling antibodies on RRs-NPs surfaces decorated with carboxyl groups deriving from PEG molecules [ 6 ], thus further highlighting the versatility of the methods. The sensitivity and specificity of the SERS nanoprobes were attested by implementing proof-of-concept experiments in biologically relevant and molecularly complex substrates such as avidin-streptavidin sandwiches and in vitro cell cultures [ 6 ].…”

“…SERS nanotags can provide selective biological tags by simply assembling antibodies on RRs-NPs surfaces decorated with carboxyl groups deriving from PEG molecules [ 6 ], thus further highlighting the versatility of the methods. The sensitivity and specificity of the SERS nanoprobes were attested by implementing proof-of-concept experiments in biologically relevant and molecularly complex substrates such as avidin-streptavidin sandwiches and in vitro cell cultures [ 6 ]. Finally, the proposed method potentially enables the synthesis of a larger set of tags, hence providing a straightforward method for custom multicolor palette for application in SERS-based multiplexing analyses.…”