## 3 Results and Discussion
### Functional Assessment of Immobilised Con A  
The fluorescence intensity measurements show that the immobilised Con A was able to bind and retain both the Alexa 488 and the FITC dextran. Moreover, it was observed (Figure 6a) that the fluorescence intensity was distributed over the ROI before glucose was added. A control experiment in which the immobilisation protocol was followed except for the addition of Con A (omitting step 3, Table 1), shows that the fluorescent dextran did not bind to the surface in any unspecific manner (Figure 6f). This suggests that the presence of Con A was the main contributing factor responsible for binding dextran to the sample surface. The fluorescence intensity decreased following incubation in MOPS buffer with increasing glucose concentration (Figure 6a-e). This result was as expected since a higher glucose concentration would be more effective in displacing bound dextran molecules, resulting in a lower concentration of the latter. This result does also agree with those obtained from the AFM measurements. Figure 5: Atomic force microscopy (AFM) image of the sample (ID7) incubated with (**a**) Alexa 488 dextran, and (**b**) Alexa 488 dextran with 40 mM glucose. It was observed that the fluorescence intensity was not evenly distributed over the sample surface. A reason for this may be that the concentration of Con A was uneven following the immobilisation step, and which was masked out by the higher fluorescent intensities at low glucose concentrations. A more uniform distribution of the protein could in principle be achieved by ensuring that the sample is 100% clean, resulting in a perfect hydroxylation step and subsequent coverage of silane. Protein aggregation could then be reduced by agitating the solution during the Con A immobilization step (e.g., by ultrasound). However, variations will still be visible if there is a loss or reduction in the binding ability of the lectin. The AFM images revealed a range in height differences on the topograph of immobilised Con A (Figures 4 and 5). This suggests that the activity of the sugar binding moieties of the lectin could also be dependent on its distance from the surface. [PERSON] [33] showed that the dissociation constant (\(K_{D}\)) for glucose was reduced from 344 \(\upmu\)M in free solution to 41 \(\upmu\)M with the lectin attached in direct contact with the surface. Since dextran is de facto long chained glucose molecules, the observed changes in the binding affinity between Con A and glucose, depending on the immobilization, are expected to yield similar changes in dextran binding to Con A. In this case the Con A-dextran complexes closest to the surface would be the ones that would dissociate last as the glucose concentration increased. Unfortunately, any cross correlation with the AFM images was not possible due to the higher resolution of the latter. Creating a more uniform distance from the substrate could be realized using specific molecular spacers that provide a minimum level of separation [33]. A third factor could be that some of the binding sites of Con A could be blocked by the surface due to different orientation of the immobilised lectin. Closer investigation of the Con A structure performed by [PERSON] [41] revealed that the surface of the molecule is dominated by two large \(\beta\)-sheets in which one constitutes the front end and one the back end. The difference lies in the front end containing the carbohydrate binding site with one hydrophilic and one hydrophobic  
Figure 6: Single fluorescence intensity images of a sample (ID6, Table 1) following Con A immobilisation, and Alexa 488 dextran incubation. The image intensity changed by immersing the sample (ID7, Table 1) with a 3-(N-morpholino) propanesulfonic acid (MOPS) buffer containing different concentrations of glucose, depicted in image series (**a**) to (**e**). The reference sample without Con A immobilisation is shown in (**f**). Image size 1.3 \(\times\) 1.7 mm\({}^{2}\). The scale bar corresponds to 250 \(\upmu\)m.  
domain.