DNA-Directed Protein Anchoring on Oligo/Alkanethiol-Coated Gold Nanoparticles: A Versatile Platform for Biosensing Applications

• Published in: Nanomaterials (Basel, Switzerland) Switzerland), 2022-12, Vol.13 (1), p.78
• Main Authors: Alsadig, Ahmed, Abbasgholi-Na, Behnaz, Vondracek, Hendrik, Medagli, Barbara, Fortuna, Sara, Posocco, Paola, Parisse, Pietro, Cabrera, Humberto, Casalis, Loredana
• Format: Article
• Language: English
• Subjects: Alkanes; Alkanethiolates; Analysis; Antigens; biosensing; Biosensors; Cancer; circulating disease biomarkers; Conjugates; Deoxyribonucleic acid; DNA; DNA-directed immobilization (DDI); Electrophoresis; ErbB-2 protein; Ethylene glycol; Frame design; Gel electrophoresis; Gold; Gold compounds; gold nanoparticles (AuNPs); Hybridization; Identification and classification; Immobilization; Mechanical properties; Methods; Microscopy; Nanobodies; Nanoparticles; Optical properties; Physicochemical properties; plasmonic immunoassays; Proteins; Self-assembled monolayers; Self-assembly; Sensitivity; Single-stranded DNA; Spectrometry; Surface plasmon resonance; Surface stability; thermal lens spectroscopy (TLS); Thermal lensing
• ISSN: 2079-4991; 2079-4991
• DOI: 10.3390/nano13010078

Herein, we report on a smart biosensing platform that exploits gold nanoparticles (AuNPs) functionalized through ssDNA self-assembled monolayers (SAM) and the DNA-directed immobilization (DDI) of DNA-protein conjugates; a novel, high-sensitivity optical characterization technique based on a miniaturized gel electrophoresis chip integrated with online thermal lens spectrometry (MGEC-TLS), for the high-sensitivity detection of antigen binding events. Specifically, we characterized the physicochemical properties of 20 nm AuNPs covered with mixed SAMs of thiolated single-stranded DNA and bio-repellent molecules, referred to as top-terminated oligo-ethylene glycol (TOEG6), demonstrating high colloidal stability, optimal binder surface density, and proper hybridization capacity. Further, to explore the design in the frame of cancer-associated antigen detection, complementary ssDNA fragments conjugated with a nanobody, called C8, were loaded on the particles and employed to detect the presence of the HER2-ECD antigen in liquid. At variance with conventional surface plasmon resonance detection, MGEC-TLS characterization confirmed the capability of the assay to titrate the HER2-ECD antigen down to concentrations of 440 ng/mL. The high versatility of the directed protein-DNA conjugates immobilization through DNA hybridization on plasmonic scaffolds and coupled with the high sensitivity of the MGEC-TLS detection qualifies the proposed assay as a potential, easily operated biosensing strategy for the fast and label-free detection of disease-relevant antigens.