**1. Introduction**

198 Electropolymerization

Microarrays are becoming a common tool in biology for screening large numbers of samples. However, the relevance of such an approach depends on the reproducibility of measurements that is directly linked to the stability of the probes grafted on the chip especially when many cycles of regeneration are performed. Indeed, regeneration of microarray chips is of great interest in improving the throughput and reducing the costs. The impact of treatments performed to remove bound ligands in order to reuse the chip depends partially on the grafted probe and on the characteristics of the probe-ligand interaction. Thus DNA microarrays are considered to be stable as oligonucleotides are highly stable molecules and hybridization reaction depends very little on the conformation of the partners; so, multiple regeneration/rehybridization procedures can be carried out without major loss of signal intensity (Benters et al., 2002, Donhauser et al., 2009). Stability of the probes is much more difficult to achieve when proteins are used. Indeed, such molecules are very complex and heterogeneous, thus there are no general rules to account for their behaviour upon different regeneration steps. Moreover, protein-protein interactions are highly susceptible to partner conformation. This is particularly true in the case of antigen (Ag) – antibody (Ab) binding, and several papers mention a loss of signal after the second or third regeneration step (Barton et al., 2008, Yakovleva et al., 2003). Peptide microarrays are a good alternative, as peptides are shorter and their stability less dependant on their tridimensional structure. Thus they are often used for antibody profiling (Cherif et al., 2006, Halperin et al., 2011, Neuman de Vegvar et al., 2003). However, there is no study dealing with the stability of such microarrays during a large samples screening (>20). Furthermore, a conformational change is not the only parameter which can impact on ligand binding. In this chapter, our aim was to analyze the evolution of the signals during samples screening and to determine which parameters are involved in the decay of the chip efficiency: grafting method, saturation step, probe itself or probe-ligand interactions, presence of protease activity in the sample. We use a microarray system based on pyrrole electropolymerization

Stability of Peptide in Microarrays: A Challenge for High-Throughput Screening 201

France) and Dr P. Morand (Centre Hospitalo-Universitaire, Grenoble, France). Serums were

Glass prisms coated with a 50 nm gold layer were obtained from Genoptics-HORIBA Scientific (Chilly-Mazarin, France). Electrodeposition was performed using an Omnigrid Micro robotic arrayer (Genoptics-HORIBA Scientific). Surface Plasmon Resonance (SPR) signals were monitored using a surface plasmon resonance imager (SPRi-Plex from Genoptics-HORIBA Scientific). Measurements were performed using SPRi dedicated software (Genoptics-HORIBA Scientific). Sample injections were ensured by a 231XL

Peptides (100µM) were grafted at least in triplicat on the gold surface of the biochip by electrochemical copolymerization of pyrrole-peptide conjugates using a solution containing 20 mmol/L pyrrole, 100 µmol/L of pyrrolated peptides and 10% glycerol in phosphate buffer (50 mmol/L). The polymerisation step was performed by a short 100 ms electrical pulse (2 V) between the counter electrode located in the needle of the microarrayer and the prism gold layer (Cherif et al., 2006, Villiers et al., 2009). Another grafting method based on electro-deposition of diazonium-peptide adducts (Corgier et al., 2009) was also used, as indicated in the text. The prism was rinsed with distilled water and saturated at room temperature for 2h using various mediums as indicated in the text. After washing with

All reactions were carried out at room temperature, in phosphate-buffered saline (PBS)/0.01%Tween 20. The flow rate in the chamber was 37 µL/min. Reflectivity was measured at 810 nm, at a fixed incidence angle (55°<<56°). After injection of serum (500 µL, 1/50 or 1/200 as indicated in the text), the biochip surface was rinsed with running buffer (10 min) to remove unbound ligands and specific binding was quantified by measuring the change in reflectivity (R) obtained after 10 min washing. The chip was regenerated using 0.1 M HCl-Glycine (pH 2.3) solution for 10 min and stabilized in the running buffer (10 min). Every twelve injections, a cleaning step was performed by injection of 1% SDS (sodium dodecyl sulphate) in water for 10 min followed by running buffer for 20 min. Saturation with NIS (1/25 in PBS), PLL-PEG or PVP as indicated in the text was realized after each

**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

sampling injector coupled to a 832 temperature regulator (Gilson, Roissy, France).

distilled water, the prism was positioned in the SPRi-Plex and used immediately.

stored at -20°C.

**2.2 Materials** 

**2.3 Peptide immobilization on gold** 

**2.4 SPRi interaction monitoring** 

**3. Results and discussion** 

cleaning step.

Fig. 1.

for probe immobilization and surface plasmon resonance imaging (SPRi) for ligand detection. The biological model consists in antigen-antibody interactions, where probes are peptides used as antigens and ligands are antibodies (Ab) contained in serum samples. Our data suggest that modification of the peptide conformation is the main parameter involved in the decrease of the signal observed upon successive uses of the chip. This conformational change leads to both a progressive reduction of the signal due to a decrease of the peptide reactivity with Ab, and the selection of Ab with the highest affinity. This phenomenon can be evaluated and must be taken into account in the analysis of the data resulting from samples screening.
