## 3 Results and Discussion  
### Structural Characterisation Using AFM  
The immobilisation of silane resulted in only a marginal difference in surface topography (Figure 2a,b), which is most likely due to the carboxyl silane forming an equally smooth surface as the unmodified quartz. The occurrence of white spots are most likely the result of contamination from the silanisation step. In contrast, the surface topography scanned after the Con A immobilisation (Figure 2c) showed densely packed small globules. Zooming in on the 3D topographic image of the protein immobilised surface (Figure 3) revealed clustered structures of one, two, three and four units, which may be representative for Con A in its monomer, dimer, trimer and tetramer state. The lectin can adapt these quaternary states depending on the pH of the solution. However, most of the clusters seem to be dimers, which conforms to the most likely state that Con A exists in when subject to a pH from 4.5-5.6 [21]. This was also the region of pH used in the immobilisation protocol of this study (pH 5.5). \begin{table}
\begin{tabular}{c l c c c} \hline \hline
**Sample ID** & **Description** & **Duration (min)** & **Number of Scans** & **Roughness,** \\ \hline
1 & Substrate samples (quartz) after dicing and & & & \\ cleaning (acetone, ethanol and DI water) & 20 & 6 & 0.12 \(\pm\) 0.04 \\
2 & Hydroxylation and silanisation step & 60 & 4 & 0.15 \(\pm\) 0.05 \\
3 & Con A immobilisation step & 120 & 10 & 0.60 \(\pm\) 0.16 \\
4 & FITC-dextran incubation & 30 & 8 & 1.14 \(\pm\) 0.35 \\
5 & FITC-dextran incubation (\(c_{glu}\) = 40 mM) & 30 & 10 & 1.12 \(\pm\) 0.50 \\
6 & Alexa-dextran incubation & 30 & 8 & 0.77 \(\pm\) 0.05 \\
7 & Alexa-dextran incubation (\(c_{glu}\) = 40 mM) & 30 & 11 & 0.76 \(\pm\) 0.22 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Sample ID notation, process preparation, and average roughness of the atomic force microscopy (AFM) image. FITC: fluorescein isothicarane. Figure 2: Atomic force microscopy (AFM) images from the immobilisation process. (**a**) Native SiO\({}_{2}\) sample, (ID1) (**b**) after silanisation, (ID2) and (**c**) after Con A immobilisation (ID3). AFM images of the samples before and after incubation with MOPS buffer containing \(c_{glu}\) = 40 mM are presented in Figures 4 and 5 The samples that were initially incubated with the smaller Alexa 488 dextran show a more evenly distributed surface compared to samples incubated with the larger FITC dextran. This result is expected if the surface roughness is scaled to the molecular size. The topographical images of the sample incubated with FITC dextran (Figure 4a,b) show peaks that are up to approximately 8 nm in height. This is larger than the 4 nm height observed with the sample incubated with Alexa 488 dextran (Figure 5a,b). These results are comparable to published data on the hydrodynamic diameter of FITC dextran (M\({}_{\text{W}}\) = 70 kDa) D\({}_{\text{F}}\) = 10.2 \(\pm\) 1.4 nm [40] and Alexa 488 dextran (M\({}_{\text{W}}\) = 3 kDa) D\({}_{\text{A}}\) = 2.33 \(\pm\) 0.38 nm [40], but smaller than the total height of Con A and dextran. This could be due to protein dehydration during preparation of these samples for the AFM studies (measured in dry state), while the published diameter is hydrodynamic. Other reasons could be that the lowest point detected by the AFM needle may not be the quartz substrate, or that the total height depends on the binding position of dextran to Con A. The number of peaks are reduced after incubating the samples with \(c_{glu}\) = 40 mM, due to the displacement of the long chained dextran with the smaller glucose molecules. Figure 4: Atomic force microscopy (AFM) image of the sample (ID5) incubated with (**a**) Fluorescein isothioxane (FITC) dextran, and (**b**) FITC dextran with 40 mM glucose. Figure 3: 3D rendered atomic force microscopy (AFM) image after Con A immobilisation, sample ID3. All units in nm.