Discrimination between protein glycoforms using lectin-functionalised gold nanoparticles as signal enhancers

Glycoforms (and other post-translational modifications) of otherwise identical proteins can indicate pathogenesis/disease state and hence new tools to detect and sense a protein's glycosylation status are essential. Antibody-based assays against specific protein sequences do not typically discriminate between glycoforms. Here we demonstrate a ‘sandwich’ bio-assay approach, whereby antibodies immobilised onto biolayer interferometry sensors first select proteins, and then the specific glycoform is identified using gold nanoparticles functionalised with lectins which provide signal enhancement. The nanoparticles significantly enhance the signal relative to lectins alone, allowing glycoform specific detection as low as 0.04 μg mL−1 (1.4 nM) in buffer, and crucially there is no need for an enrichment step and all steps can be automated. Proof of concept is demonstrated using prostate specific antigen: a biomarker for prostate cancer, where glycoform analysis could distinguish between cancerous and non-cancerous status, rather than only detecting overall protein concentration.

Working solutions of anti-PSA Ab, PSA and AuNPs@lectins were prepared in 10 mM HEPES buffer pH 7.5 with 100 mM NaCl, 0.1 mM CaCl2, 0.01 mM MgCl2. EDC/sulfo-NHS solution was freshly prepared every day, before the experiment begins, in acetate buffer pH 5.0. Ultrapure water (resistivity =18.2MΩ.cm (at 25 °C)) was used throughout this work. All chemicals employed were of analytical reagent grade.

Gold nanoparticles (AuNPs) synthesis
35 nm gold nanoparticles (AuNPs) were synthesised following a method described elsewhere. was again injected. After 20 min, the reaction was finished, and this process was repeated twice.
Afterwards, the sample was diluted by adding 85 mL of MilliQ water and 3.1 mL of 60 mM sodium citrate. This solution was then used as a seed solution, and three further portions of 1.6 mL of 25 mM HAuCl4 were added with 20 min between each addition. Following completion S4 of this step 1 mL was taken for DLS and UV/Vis analysis. The sample was diluted by adding 135 mL of MilliQ water and 4.9 mL of 60 mM sodium citrate. This solution was then used as a seed solution, and the process was repeated with three further additions of 2.5 mL of 25 mM HAuCl4, this solution was analysed by DLS and UV/Vis and target size of 35 nm was reached and the solution was cooled and a sample taken for TEM analysis.

AuNPs functionalisation with lectins
The AuNPs were coated with the lectins SNA, MAL I, WFL (final concentration 50 µg.mL -1 ) in a reproducible and stable manner for 7 hours, with gentle stirring at room temperature. After a centrifugation step (4500 rpm) for 20 min the supernatant was removed, eliminating the unbound protein, and the Lectin@AuNP conjugates pellet was re-dispersed in 850 μL of HEPES buffer pH 7.5.

Dynamic Light Scattering (DLS)
Hydrodynamic diameters (Dh) and size distributions of particles were determined by DLS using a Malvern Zetasizer Nano ZS with a 4 mW He-Ne 633 nm laser module operating at 25 ℃. Measurements were carried out at an angle of 173° (back scattering), and results were analysed using Malvern DTS 7.03 software. All determinations were repeated 5 times with at least 10 measurements recorded for each run. Dh values were calculated using the Stokes-Einstein equation where particles are assumed to be spherical.

Transmission Electron Microscopy (TEM)
Dry-state stained TEM imaging was performed on a JEOL JEM-2100Plus microscope operating at an acceleration voltage of 200 kV. All dry-state samples were diluted with deionised water and then deposited onto formvar-coated copper grids.

X-Ray Photoelectron Spectroscopy (XPS)
The samples were attached to electrically-conductive carbon tape, mounted on to a sample bar and loaded in to a Kratos Axis Ultra DLD spectrometer which possesses a base pressure below 1 x 10 -10 mbar. XPS measurements were performed in the main analysis chamber, with the sample being illuminated using a monochromated Al Kα x-ray source. The measurements were S5 conducted at room temperature and at a take-off angle of 90° with respect to the surface parallel. The core level spectra were recorded using a pass energy of 20 eV (resolution approx.

UV-vis Spectroscopy
Absorbance measurements were recorded on a BioTek Epoch Microplate Reader using 96-well plates purchased from Greiner Bio-one.
All bioassays were performed in black 96-well plates acquired from Greiner Bio-one. The optimised BLI-bioassay (illustrated in Figure S1) was setup and preformed according to the following procedure: Amine reactive (ARG2) biosensor tips, previously hydrated in milliQ H2O water were (1) dipped in milliQ water for 1 minute to ensure that a stable baseline was established. Afterwards, the biosensors were first activated using (2)

AuNPs characterisation
The citrate-stabilised gold nanoparticles were also characterised by DLS and TEM analysis.
Example images and histogram analyses of colloidally stable particles are shown below.

Analysed XPS data
Lectin coating was further confirmed by use of XPS. In particular, the presence of amides [from N 1s], that are not present on the naked particles, confirmed successful binding of the lectins onto the particle surface.  Figure