Fabrication of Highly Stable Glyco‐Gold Nanoparticles and Development of a Glyco‐Gold Nanoparticle‐Based Oriented Immobilized Antibody Microarray for Lectin (GOAL) Assay

Abstract

The design of high-affinity lectin ligands is critical for enhancing the inherently weak binding affinities of monomeric carbohydrates to their binding proteins. Glyco-gold nanoparticles (glyco-AuNPs) are promising multivalent glycan displays that can confer significantly improved functional affinity of glyco-AuNPs to proteins. Here, AuNPs are functionalized with several different carbohydrates to profile lectin affinities. We demonstrate that AuNPs functionalized with mixed thiolated ligands comprising glycan (70 mol %) and an amphiphilic linker (30 mol %) provide long-term stability in solutions containing high concentrations of salts and proteins, with no evidence of nonspecific protein adsorption. These highly stable glyco-AuNPs enable the detection of model plant lectins such as Concanavalin A, wheat germ agglutinin, and Ricinus communis Agglutinin 120, at subnanomolar and low picomolar levels through UV/Vis spectrophotometry and dynamic light scattering, respectively. Moreover, we develop in situ glyco-AuNPs-based agglutination on an oriented immobilized antibody microarray, which permits highly sensitive lectin sensing with the naked eye. In addition, this microarray is capable of detecting lectins presented individually, in other environmental settings, or in a mixture of samples. These results indicate that glyconanoparticles represent a versatile and highly sensitive method for detecting and probing the binding of glycan to proteins, with significant implications for the construction of a variety of platforms for the development of glyconanoparticle-based biosensors.

Current applications of antibody microarrays

“…Hu et al [ 107 ] developed a hierarchically nanostructured organic–inorganic hybrid substrate, comprising of randomly oriented ZnO nanorods on glass slides with coaxially tethered dense polymer brush, which highly improved the limit of detection (LOD) to 100 fg/mL. Huang et al [ 108 ] have invented a glycol-gold nanoparticle-based antibody microarray, with potential to amplify a signal that could be detected by the naked eye.…”

Glyco-gold nanoparticles: synthesis and applications

“…Based on the LSPR phenomenon, the controlled aggregation and de-assembling of AuNPs is reflected in a detectable variation of the colloidal solution color. Therefore, several biosensors for the detection of proteins [42], lectin [25,27,43], cancer biomarkers [28] and viruses [44–45] were developed. Photoluminescence properties of ultrasmall gold nanoclusters and nanodots have been exploited to detect Escherichia coli (a bacteria containing mannose-binding receptors) [46] and thyroglobulin in serum [47] and to study the internalization pathway in dendritic cells, employing confocal laser microscopy (CLSM) and flow cytometry [48].…”

“…Generally, the majority of the spacers are thiol-linkers, exploiting the strong soft–soft interaction between gold and sulfur. The spacer has the crucial role to stabilize GAuNPs in the surrounding media, which is fundamental in the case of colorimetric bioassays where the NP colloidal stability can strongly influence the state of aggregation and, consequently, the analysis results [43,65–66]. Woods et al, in a recent paper, performed a computational study to evaluate the role of the spacer in the glycan–protein interaction [67].…”

“…A very interesting result reports on the design of highly stable carbohydrate-coated gold nanoparticles, able to recognize at subnanomolar and low picomolar concentrations level of ConA, wheat germ agglutinin and Ricinus communis agglutinin [43]. In this case, a careful decoration of the gold surface has been carried out by employing a thio-amphiphilic linker to impart improved solubility and flexibility to the glycosyl-ligand.…”

Nanotechnologies in Protein Microarrays

“…The most frequent applications are studying of lectins affinity to carbohydrate substrates [52]. New chip-based method to detect protein glycosylation based on the energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs) was developed by Kim et al [53].…”