# Design of Antibody-Functionalized Polymeric Membranes for the Immunoisolation of Pancreatic Islets
## 3 Results and Discussion
### GFP Immobilization  
The NTA-lysine-Ni\({}^{2+}\)-modified surfaces have been widely used to target hexahistidine (His6) proteins [34]. Indeed, oligo-histidine (His) sequences in combination with NTA-lysine-Ni\({}^{2+}\) are employed for purification through chromatographic techniques since the binding is highly selective as well as reversible [35; 36]. In this study, we exploited this coordination chemistry to bind the histidine residues of the His-tagged GFP to NTA-lysine-Ni\({}^{2+}\) modified surfaces in a reversible manner.  
To estimate the amount of protein attached to the substrate, it was exposed to imidazole buffer. Indeed, the addition of a competitive ligand, such as histidine or imidazole, or a chelating agent, such as EDTA, enables the triggered release of His-tagged ligand from the surface [37]. Remarkably, such a reversible functionalization results in the reusability of the functionalized supports for successive experiments. The detached GFP, named \"eluted GFP\", was quantified by both fluorescence analysis and Western Blot.  
In the case of the fluorescence analysis, the curve of the eluted protein was compared to the recovered fraction (that corresponds to the unbound protein amount) and to the feeding solution of GFP. The plot of the fluorescent intensities is reported in Figure 5a.  
As shown in Panel (b) of Figure 5 the decrease in fluorescence intensity of recovered GFP (dark green bar) when compared to the feeding solution (light green bar) was statistically significant (\(p<0.001\)), thus indicating that about 50% of the protein was immobilized onto the surface [38].  
Figure 5: (a) Fluorescence emission spectra and relative intensity percentage of green fluorescent protein (GFP) solutions of solutions (excitation 395 nm, emission 508 nm) as quantitative estimation of the protein recovered after incubation (dark green curve) and eluted from the solid support (red curve) compared to the feeding solution of GFP (light green curve) (**b**). The quantitative values have been averaged over 3 measurements. Error bars represent standard deviation (SD) (*** \(p<0.001\), ** \(p<0.01\)).  
However, the fluorescence intensity at 508 nm of the eluted GFP (red bar) had a relative intensity of about 8.4%. To explain this discrepancy, it is plausible that not all the protein was detached and eluted, and it is likely that a fraction of the eluted GFP could be quenched. Fluorescence quenching may occur when the fluorophore is partially denaturated or because it is unprotected from quenching by jostling water dipoles, paramagnetic oxygen molecules, or cis-trans isomerization [39; 40]. Moreover, it is reported that wild-type GFP is influenced by pH variations; indeed, it is quenched by acidic pH values, whereas at basic pH, it is absorbance and excitation amplitude decreases [41; 42; 43].  
To confirm these findings, the fluorescence estimation of GFP was compared to the densitometry analysis of the Western Blots (WB) bands [44]. Figure 6a shows the WB analysis of the three fractions, whereas in Figure 6b the densitometric values are reported as percentages of the control values.  
Quantification of the eluted GFP by WB indicates that about 13.5% of initial GFP protein was detached from the substrate. These results are comparable to those from the fluorescence analysis, and confirm that imidazole 500 mM seemed to be not able to elute all the immobilized GFP and that GFP could be partially quenched [41]. The estimation of the protein density per surface unit was equal to 54 nmol cm\({}^{-2}\).