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

Abstract

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.

Recent Progresses in Plasmonic Biosensors for Point-of-Care (POC) Devices: A Critical Review

“…On this basis, engineering Nbs in such a way as to realize an optimal electrostatic configuration by integrating strong binding residues, such as cysteine, and then creating Nb-AuNP colloids through physical absorption has been proposed as a general strategy for the development of a successful sensing system. One of the early approaches in the use of AuNPs conjugated with nanobodies as a sensing device was presented in the work of Alsadig [49]: an advanced biosensing platform was created through a combination of a mixed self-assembled monolayer (SAM) of thiolated single-stranded DNA (ssDNA) and bio-repellent molecules, indicated as top-terminated oligo-ethylene glycol (TOEG6) and the DNA-directed immobilization (DDI) of DNA-protein conjugates. One of the primary challenges related to the immobilization of protein onto a solid substrate concerns the preservation of the protein functionality by preventing alterations in their structure during the immobilization progress, along with the steric hindrance of the recognition sites.…”

DNA Sensing Platforms: Novel Insights into Molecular Grafting Using Low Perturbative AFM Imaging

“…Nanografting was initially applied to alkanethiol SAMs [33,38], and has been successfully exploited to produce DNA nano/micropatterns in alkanethiol SAMs [20,21,[39][40][41][42][43][44]. Selective hybridization of the nanografted regions with complementary DNA has been used to anchor functional peptides/proteins to the nanografted platforms by exploiting DNA-directed immobilization [22][23][24]45].…”