Polymer-tethered glyconanoparticle colourimetric biosensors for lectin binding: structural and experimental parameters to ensure a robust output

Glycan–lectin interactions play essential roles in biology; as the site of attachment for pathogens, cell–cell communication, and as crucial players in the immune system. Identifying if a new glycan (natural or unnatural) binds a protein partner, or if a new protein (or mutant) binds a glycan remains a non-trivial problem, with few accessible or low-cost tools available. Micro-arrays allow for the interrogation of 100's of glycans but are not widely available in individual laboratories. Biophysical techniques such as isothermal titration calorimetry, surface plasmon resonance spectrometry, biolayer interferometry and nuclear magnetic resonance spectroscopy all provide detailed understanding of glycan binding but are relatively expensive. Glycosylated plasmonic nanoparticles based on gold cores with polymeric tethers have emerged as biosensors to detect glycan–protein binding, based on colourimetric (red to blue) outputs which can be easily interpreted by a simple UV-visible spectrometer or by eye. Despite the large number of reports there are no standard protocols for each system or recommended start points, to allow a new user to deploy this technology. Here we explore the key parameters of nanoparticle size, polymeric tether length and gold concentration to provide some guidelines for how polymer-tethered glycosylated gold nanoparticles can be used to probe a new glycan/protein interactions, with minimal optimisation barriers. This work aimed to remove the need to explore chemical and nanoparticle space and hence remove a barrier for other users when deploying this system. We show that the concentration of the gold core is crucial to balance strong responses versus false positives and recommend a gold core size and polymer tether length which balances sufficient colloidal stability and output. Whilst subtle differences between glycans/lectins will impact the outcomes, these parameters should enable a lab user to quickly evaluate binding using minimal quantities of the glycan and lectin, to select candidates for further study.


Mass spectrometry
Low resolution mass spectra (LRMS) were recorded on a Bruker Esquire 2000 spectrometer using electrospray ionisation (ESI). M/z values are reported in Daltons.

FT-IR Spectroscopy
Fourier Transform-Infrared (FT-IR) spectroscopy measurements were carried out using an Agilent Cary 630 FT-IR spectrometer, in the range of 650 to 4000 cm -1 .

Size Exclusion Chromatography
Size exclusion chromatography (SEC) analysis was performed on an Agilent Infinity II MDS instrument equipped with differential refractive index (DRI), viscometry (VS), dual angle light scatter (LS) and variable wavelength UV detectors. The system was equipped with 2 x PLgel Mixed D columns (300 x 7.5 mm) and a PLgel 5 µm guard column. The mobile phase used was DMF (HPLC grade) containing 5 mM NH4BF4 at 50 ℃ at flow rate of 1.0 mL.min -1 .
Poly(methyl methacrylate) (PMMA) standards (Agilent EasyVials) were used for calibration between 955,000 -550 g.mol -1 . Analyte samples were filtered through a nylon membrane with 0.22 μm pore size before injection. Number average molecular weights (Mn), weight average S5 molecular weights (Mw) and dispersities (ĐM = Mw/Mn) were determined by conventional calibration and universal calibration using Agilent GPC/SEC software.

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 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. 0.4 eV), from an analysis area of 300 μm x 700 μm. The spectrometer work function and binding energy scale of the spectrometer were calibrated using the Fermi edge and 3d5/2 peak recorded from a polycrystalline Ag sample prior to the commencement of the experiments. In order to prevent surface charging the surface was flooded with a beam of low energy electrons throughout the experiment and this necessitated recalibration of the binding energy scale. To achieve this, the C-C/C-H component of the C 1s spectrum was referenced to 285.0 eV. The data were analysed in the CasaXPS package, using Shirley backgrounds and mixed Gaussian-Lorentzian (Voigt) lineshapes. For compositional analysis, the analyser transmission function has been determined using clean metallic foils to determine the detection efficiency across the full binding energy range.

Dynamic Light Scattering
Hydrodynamic diameters (Dh) and size distributions of particles were determined by dynamic light scattering (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 S6 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.

UV-vis Spectroscopy
Absorbance measurements were recorded on an Agilent Cary 60 UV-Vis Spectrophotometer and on a BioTek Epoch microplate reader.

Transmission Electron Microscopy
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 deionized water and then deposited onto formvar-coated copper grids.

Materials
All chemicals were used as supplied unless otherwise stated. Distilled water used for buffers was MilliQ grade 18.2 mΩ resistance.

Additional Data and Figures
Polymer Characterization Figure S1. Normalised size exclusion chromatography RI molecular weight distributions of telechelic PHEA obtained in DMF verses PMMA standards.

Nanoparticle Characterization
The citrate-stabilized gold nanoparticles were characterized by DLS, UV/VIS and TEM.
Example images and histogram analyses (from >100 particles) are shown below.        Gal-pHEA40@AuNP56 6.25 56 Table S3. Estimate of limit detection of the AuNP-pHEA systems for SBA. This is not intended as a limit for e.g. diagnostics but to show how each formulation can give a different response and why the polymer linker length is crucial.