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. Graphical abstract.

Keywords: Kinetics; Localized surface plasmon resonance (LSPR); Protein immobilization; Quantitative interaction analysis; Real-time biosensor; Self-assembly.

Graphical abstract

Fig. 1

Self-assembly of functionalized AuNP monolayers for LSPR detection of protein interactions. a Molecular structure of PLL-PEG-OPSS for coating the substrate surface. b Schematic illustration of an AuNP monolayer on a PLL-PEG-OPSS-coated glass slide. c Molecular structure of trisNTA-OEG-SS with bound Ni²⁺ ions for reversible immobilization of His-tagged proteins. d Surface functionalization of AuNP monolayers for site-specific protein immobilization via functional thiols. e Scheme of multivalent hexahistidine-tagged protein binding on a trisNTA-OEG-SS-functionalized AuNP

Fig. 2

Self-assembly of 40 nm AuNP monolayers on PLL-PEG-OPSS-coated surfaces. a Formation of a stable PLL-PEG-OPSS polymer coating on a silica substrate monitored by reflectance interference spectroscopy (RIfS). b Real-time RIfS binding curve of 6-fold concentrated AuNPs to PLL-PEG-OPSS-coated silica substrate. c AFM image of the AuNP monolayer in buffer solution

Fig. 3

Responsiveness of AuNP monolayers quantified by LSPR reflectance spectroscopy. a, b Full reflectance spectra a and zoom into the peak reflectivity b of an AuNP monolayer assembled on a PLL-PEG-OPSS-coated glass slide upon injecting glucose at different concentrations. Glucose concentrations are indicated in the legend of panel B. c Linear correlation of the relative reflectivity determined at the peak of the reflectance spectra with the glucose concentration. The relative reflectivity was determined by subtracting the reflectivity in the absence of glucose. LSPR chips were prepared from 6- (blue) or 10-fold (black) concentrated AuNP solutions

Fig. 4

Specific and reversible immobilization of His-tagged proteins onto AuNP monolayers functionalized with trisNTA-OEG-SS. a Cartoon depicting reversible immobilization of H6-mEGFP. b Typical binding assay detected by LSPR spectroscopy including the following sample injections: (i) 250 mM EDTA, (ii) 10 mM NiCl₂, (iii) 500 mM imidazole, (iv) 1 μM H6-mEGFP, and (v) 500 mM imidazole. c Immobilization of H6-mEGFP repeated twice on the same LSPR chip and control experiments using mEGFP without His-tag (dashed black line), or H6-mEGFP without Ni²⁺ ions (black line). d Scheme of capturing tagless mEGFP via H10-HaloTag-NB immobilized on a trisNTA-functionalized LSPR chip. e On the Ni²⁺-ion conditioned AuNP monolayer, injections are (i) 300 nM H10-HaloTag-NB, and (ii) 100 nM of tagless mEGFP

Fig. 5

Reversible protein–protein interaction analysis by LSPR reflectance spectroscopy. a Cartoon of the assay. Immobilization of IFNAR2-H10 (blue, i), reversible binding of IFNα2 (red, ii) and surface regeneration by imidazole (iii) on a trisNTA-functionalized AuNP monolayer. b Changes in reflectivity upon injection of 500 nM IFNAR2-H10 (i), 500 nM IFNα2 (ii), and 500 mM imidazole (iii). c–e Dependency of ligand binding to receptor densities quantified by LSPR reflectance spectroscopy. c Alternation of receptor surface densities on a trisNTA-functionalized AuNP monolayer by injecting different concentrations of IFNAR2-H10 (i), followed by an injection of 1 μM MBP-H10 (ia). d LSPR signals of binding 500 nM IFNα2 onto immobilized IFNAR2-H10 with different densities. Color coding of curves is the same as in panel c. e Plots of relative LSPR reflectivity amplitudes of bound IFNα2 versus immobilized IFNAR2-H10. The red line shows linear regression yielding a slope of 0.49 ± 0.02 and an intercept 0.003 ± 0.0001

Fig. 6

Quantification of the IFNα2-IFNAR2 interaction kinetics by LSPR reflectance spectroscopy. a Normalized IFNα2 binding curves obtained for different densities of immobilized IFNAR2-H10. Concentrations of IFNAR2-H10 used for immobilization are indicated by the legend. b Fitting of association and dissociation phases (red lines), respectively, of the IFNα2 binding curve obtained upon immobilization of IFNAR2-H10 at 20 nM