**3.2 What can influence grafted peptide stability?**

SPR signal is directly related to probe-ligand binding which depends on many parameters at both probe and ligand levels. Various hypotheses are summarized in Fig. 2.

SPR signal depends on the quantity and the quality of both probe and ligand. In our experiments, ligands were Ab from a rabbit anti-serum which were aliquoted and kept at 8°C in the autoinjector rack. Thus it is unlikely that any changes in the Ab content of the samples occur.

The problem is more complex when we consider the probe. First, the decrease of the binding capacity could be due to a reduction in the number of available binding sites (epitopes) for the injected Ab during the set of injection/regeneration cycles. This could be due to either a gradual release of the grafted peptide from the chip surface or a proteolysis of the peptide due to the presence of proteolytic activity in the injected samples. The quality of the probe is also an important parameter for Ab binding as it must have a proper conformation and a good accessibility. Thus, if the regeneration step is partially inefficient, remaining Ab may

Fig. 1. Peptide chip stability. Eight independent experiments were realized as follows: C131 was immobilized in triplicate on the gold chip surface via pyrrole electropolymerization. Successive injections of non immune serum (1/50) were performed, followed by HCl-Glycine regeneration. Anti-C131 serum (1/200) was periodically injected (every 12 injections) and SPR signal was quantified. Results were standardized according to the

We observed a progressive decrease in the SPR signal during the experiments. This decay is very reproducible from one experiment to another and presents a biphasic profile with a rapid drop during the first 12 injections followed by a slighter decrease upon the subsequent injections. Such a loss of efficiency could limit the use of peptide chip for samples screening, therefore it is important to identify the parameters involved in this phenomenon to limit its

SPR signal is directly related to probe-ligand binding which depends on many parameters at

SPR signal depends on the quantity and the quality of both probe and ligand. In our experiments, ligands were Ab from a rabbit anti-serum which were aliquoted and kept at 8°C in the autoinjector rack. Thus it is unlikely that any changes in the Ab content of the

The problem is more complex when we consider the probe. First, the decrease of the binding capacity could be due to a reduction in the number of available binding sites (epitopes) for the injected Ab during the set of injection/regeneration cycles. This could be due to either a gradual release of the grafted peptide from the chip surface or a proteolysis of the peptide due to the presence of proteolytic activity in the injected samples. The quality of the probe is also an important parameter for Ab binding as it must have a proper conformation and a good accessibility. Thus, if the regeneration step is partially inefficient, remaining Ab may

both probe and ligand levels. Various hypotheses are summarized in Fig. 2.

signal obtained for the first injection (100%).

**3.2 What can influence grafted peptide stability?** 

impact.

samples occur.

prevent de novo binding, either directly or indirectly by steric hindrance. Various sets of experiments were performed in order to determine which parameters are involved in the chip efficiency decay.

Fig. 2. Parameters involved in the signal loss: tree of possibilities
