Self-assembly of robust gold nanoparticle monolayer architectures for quantitative protein interaction analysis by LSPR spectroscopy

Abstract

Localized surface plasmon resonance (LSPR) detection offers highly sensitive label-free detection of biomolecular interactions. Simple and robust surface architectures compatible with real-time detection in a flow-through system are required for broad application in quantitative interaction analysis. Here, we established self-assembly of a functionalized gold nanoparticle (AuNP) monolayer on a glass substrate for stable, yet reversible immobilization of Histidine-tagged proteins. To this end, one-step coating of glass substrates with poly-L-lysine graft poly(ethylene glycol) functionalized with ortho-pyridyl disulfide (PLL-PEG-OPSS) was employed as a reactive, yet biocompatible monolayer to self-assemble AuNP into a LSPR active monolayer. Site-specific, reversible immobilization of His-tagged proteins was accomplished by coating the AuNP monolayer with tris-nitrilotriacetic acid (trisNTA) PEG disulfide. LSPR spectroscopy detection of protein binding on these biocompatible functionalized AuNP monolayers confirms high stability under various harsh analytical conditions. These features were successfully employed to demonstrate unbiased kinetic analysis of cytokine-receptor interactions. Keywords Localized surface plasmon resonance (LSPR). Self-assembly. Real-time biosensor. Protein immobilization. Quantitative interaction analysis. Kinetics Abbreviations AuNP Gold nanoparticle HaloTag-NB HaloTag fused with anti-GFP nanobody HTL HaloTag ligand IFNAR2 Type I interferon receptor subunit 2 IFNα2 I n t e r f e r o n-α2 LSPR Localized surface plasmon resonance mEGFP Monomeric enhanced green fluorescent protein PLL-PEG-OPSS Poly-L-lysine graft poly(ethylene glycol) terminated with ortho-pyridyl disulfide TrisNTA Tris-nitrilotriacetic acid Published in the topical collection Advances in Direct Optical Detection with guest editors Antje J. Baeumner, Günter Gauglitz, and Jiri Homola.

Biofunctional Nanodot Arrays in Living Cells Uncover Synergistic Co‐Condensation of Wnt Signalodroplets

“…Surface modification with 1‐napthylmethyltrichlorosilane (NMTS) turned out most efficient compared to other hydrophobic silanes (Figure S1A, Supporting Information), for which the binding kinetics of BSA was quantified by label‐free detection based on reflectance interference (RIF) under flow‐through conditions. [ 48–50 ] Very rapid and stable BSA coating was observed on NMTS‐treated silica substrates (Figure 2B; Figure S1b, Supporting Information) with the final mass signal of ≈3.5 ng mm −2 being in good agreement with formation of a BSA monolayer. [ 51 ] Specific immobilization of the mEGFP‐HaloTag fusion protein on the HTL BSA‐coated surface was confirmed by real‐time total internal reflection fluorescence (TIRF) detection (Figure 2C), with an association rate constant ( k on ) of ≈10 3 M −1 s −1 (Table S1, Supporting Information) in line with previous kinetics studies of the HaloTag‐HTL interaction.…”

“…Surface modification with 1-napthylmethyltrichlorosilane (NMTS) turned out most efficient compared to other hydrophobic silanes (Figure S1A, Supporting Information), for which the binding kinetics of BSA was quantified by label-free detection based on reflectance interference (RIF) under flow-through conditions. [48][49][50] Very rapid and stable BSA coating was observed on NMTS-treated silica substrates (Figure 2B; Figure S1b…”

Biofunctional nanodot arrays in living cells uncover synergistic co-condensation of Wnt signalodroplets

“…Surface modification with 1-napthylmethyltrichlorosilane (NMTS) turned out most efficient compared to other hydrophobic silanes (Supplementary Fig. S1A), for which the binding kinetics of BSA was quantified by label-free detection based on reflectance interference (RIF) under flow-through conditions (48)(49)(50). Very rapid and stable BSA coating was observed on NMTS-treated silica substrates (Fig.…”

Identification of Proteins Using Supramolecular Gold Nanoparticle‐Dye Sensor Arrays

“…[48] When different proteins interact with AuNPs, the local environment of both protein and AuNP are changed. [49] In addition, due to the hydrophobic interactions between AuNPs and proteins, the conformation of AuNP hydrophobic monolayer might change, [50] varying the arrangement of the dyes in the AuNP. These changes can either restore or further quench the fluorescence signals of dyes.…”