Precise Generation of Selective Surface-Confined Glycoprotein Recognition Sites

Since glycoproteins have become increasingly recognized as key players in a wide variety of disease processes, there is an increasing need for advanced affinity materials for highly selective glycoprotein binding. Herein, for the first time, a surface-initiated controlled radical polymerization is integrated with supramolecular templating and molecular imprinting to yield highly reproducible synthetic recognition sites on surfaces with dissociation constants (KD) in the low micromolar range for target glycoproteins and minimal binding to nontarget glycoproteins. Importantly, it is shown that the synthetic strategy has a remarkable ability to distinguish the glycosylated and nonglycosylated forms of the same glycoprotein, with a >5-fold difference in binding affinity. The precise control over the polymer film thickness and positioning of multiple carbohydrate receptors plays a crucial role in achieving an enhanced affinity and selectivity. The generated functional materials of unprecedented glycoprotein recognition performance open up a wealth of opportunities in the biotechnological and biomedical fields.


Dynamic Contact Angle
All contact angle measurements were taken using an Attension Theta Contact Angle Meter (Biolin Scientific). An automated microsyringe was used to inject and retract the water droplet onto and from the surfaces from which the advancing and receding angles were then measured by recording the change in the pinning angle by video (acquisition rate 35 frames per second).
OneAttension software was then used to analyse the advancing and receding angles. For each surface type (i.e. SAM or polymer), at least 3 measurements each from 3 individual chips were taken (n=9) from which the average and standard deviation values were calculated.

Ellipsometry
The thicknesses of the SAM and polymer surfaces were measured with spectroscopic ellipsometry using a Jobin-Yvon UVISEL ellipsometer with a xenon light source. The incident angle of the light was fixed at 70° for all measurements and the wavelength range was 220-800 nm. The calculation of the film thicknesses were based on a three-phase ambient atmosphere/SAM/Au model. The SAM was assigned a refractive index of 1.49 and assumed to be isotropic. The thicknesses were reported using averages of at least 3 independent surfaces with 3 measurements obtained from each surface (n=9) and the standard deviation was calculated from these values.

X-ray Photoelectron Spectroscopy (XPS)
XPS spectra were acquired using an Escalab 250, Thermo Scientific K-Alpha. The system used a monochromatic Al kα source with a photon energy of 1486.68 eV and for each measurement a spot size of 0.2 mm 2 was used with a take-off angle of 90° to the surface of the plane. All measurements were undertaken at a pressure of ~7.5 x 10 -9 Torr. The SAM samples were taped onto a stainless steel plate using Shintron tape and clipped with stainless steel clips. Each SAM S4 measurement was undertaken with the charge neutraliser on to prevent charging issues. However, for polymer samples (poly(MEBA) or poly(MEBA)-APBA)), the charge neutraliser was turned off as this prevented charging of the surfaces. The high resolution spectra were obtained using a

Atomic Force Microscopy
All AFM images were acquired using an Asylum Research MFP-3D AFM (Oxford Instruments, UK) operating in Intermittent Contact Mode at a temperature of 18 o C and a relative humidity of <40 %. Images were composed of 512 x 512 pixels and the scanning velocity was 10 μm/s. Rectangular pyramidal-tipped Si cantilevers (PPP-NCL, Windsor Scientific, UK) were employed; their nominal length, width, and tip diameter were 225 μm, 38 μm and <10 nm, respectively. Images were analysed using Scanning Probe Image Processor software (Image Metrology, Denmark). Images were then presented using Gwyddion software (Version 2.51). The Ra roughness of the modified gold surfaces were calculated using the 'Statistical Quantities' tool of Gwyddion software (Version 2.51). Here, random points across the surfaces were selected and the Ra values recorded. The average Ra roughness was calculated from 3 points each from 3 individual chips (n=9).

Surface Plasmon Resonance (SPR)
All protein SPR experiments were run on a Reichert SR7000DC Dual Channel Spectrometer (NY, USA). All SPR chips were purchased from Reichert (Depew, NY, USA). The running buffer used for all experiments was degassed 1 x PBS containing 96 mM glycine, 10 mM HEPES, and 0.01 % sodium dodecyl sulfate adjusted to pH 8.6 using potassium hydroxide. Before each S5 measurement the acidic regeneration buffer (consisting of equal parts of 75 mM malonic, phosphoric, oxalic and formic acid) was run across the surface to remove any contaminates during set up. The protein solutions were injected across the surfaces at 40 μL/min for 10 mins, following which the running buffer was used for the 15 minutes dissociation phase. The surfaces were then regenerated using the acidic regeneration buffer for 10 minutes. Data sets were analysed using Scrubber 2 (BioLogic Software, Campbell, Australia). All SPR responses at Req were plotted against the concentration of the injected proteins (Cp) and fitted to a 1:1 steady-state model using Scrubber 2. The model uses a non-linear least-squares regression method to fit data to the Langmuir adsorption isotherm (Equation S1), with KD being the dissociation constant and Rmax the maximum analyte binding capacity of the surface.
Scheme S1 -Synthetic strategy for the synthesis of 11-DTMBD.
The 11,11'-disulfanediylbis(undecan-1-ol) 2 (0.4 g, 0.98 mmol) was added to a separate, stirred solution of 30 mL THF and dry pyridine (175 μL, 2.16 mmol) and kept at 0-5 °C also under an argon atmosphere. The 10 mL solution containing the acid bromide was then slowly added dropwise to the 30 mL disulfide solution. The reaction was stirred for 2 hrs at 0-5 °C and then for a further 16 hrs at room temperature. Afterwards the reaction was diluted with DCM (60 mL) and extracted with cold 1 N HCl (2 × 100 mL), saturated NaHCO3 (1 × 100 mL) and saturated NaCl (1 × 100 mL). The organic phase was retained and all aqueous phases were back extracted with DCM (1 × 100 mL). The solvent was removed under vacuum and the crude product was purified by chromatography on silica gel (hexane/DCM, 1:1). The purified 11-DTMBD product (0.2 g, 28.9 %) was a clear, colourless oil. 1

CD of RNase B with increasing percentages (v/v) of MeOH
To begin with, a 50 μM RNase B solution was prepared using 0.

Formation of the 11-DTMBD SAMs
The gold substrates were cleaned by immersion in piranha solution (70% H2SO4, 30% H2O2) at room temperature for 10 minutes, rinsed with ultra-high quality (UHQ) water and HPLC grade S8 subsequently 1.02 µL of ethyl-2-bromoisobutyrate (E-2-BB) was injected. The substrates were polymerised for 30 minutes, during which the solution was continually degassed using a slow stream of argon, after which they were then were removed from the polymerisation solution, rinsed extensively for 3 minutes with ultra-high pure H2O, then with HPLC grade EtOH and finally dried under a stream of argon.

Formation of surface molecularly imprinted polymers (MIP) and non-imprinted polymers (NIP) from 11-DTMBD SAMs
The