**3.1 Is a grafted peptide sufficiently stable to allow a multiple re-use of the chip?**

To assess the stability of the peptide chip during samples screening, we immobilized C131 peptide using pyrrole electropolymerization and performed multiple injection/regeneration cycles using non immune serum (NIS) with periodical injections of anti-C131 serum to monitor the reactivity with the grafted probe. SDS cleaning was realized every twelve injections, as described in §2.4. The SPR (Surface Plasmon Resonance) signal obtained for each injection was monitored. Results from eight independent experiments are presented in Fig. 1.

Stability of Peptide in Microarrays: A Challenge for High-Throughput Screening 203

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

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

upon successive anti-C131 serum injections were recorded in both cases.

but this eventuality will be discussed later on in light of the other results (§ 4).

If probe release occurs, this may be related to the nature of the link between the probe and the chip surface, i.e. to the grafting process. To evaluate this hypothesis, C131 peptide was immobilized using either electropolymerization of pyrrole-peptide conjugates or electrodeposition, as a monolayer, of diazonium-peptide adducts and the signals obtained

As shown in Fig. 3, the stability of the SPR signal was much better when C131 was immobilized via diazonium, but the change in reflectivity is much lower (likely due to lower probe density on the surface), impairing the sensitivity of the system. At first sight, this observation fits with a probe release in the case of pyrrole electropolymerization protocol,

**3.3 Influence of the grafting process** 

chip efficiency decay.

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 signal obtained for the first injection (100%).

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 impact.
