**6. References**

212 Electropolymerization

To validate this correction procedure, we realised the following experiments: 14 peptides (11 from HDV, 1 from HEL, 1 from Ova and the control peptide C131) were grafted in triplicates on 2 chips. A set of 17 serums (14 from HDV infected patients and 3 from healthy donors) was injected twice on the first chip and once on the second chip. Serums were in a different random order within each set. Rabbit anti-C131 serum was periodically injected (at least every 12 injections) to establish a control curve for signal decrease. The sum of the SPR signals obtained for each peptide was calculated for each injection set, before and after application of the correction, as well as the standard deviation (Fig. 10B). Indeed, as signal loss depends on the peptide/ligand pair, this correction is not optimal, but nevertheless

Among factors susceptible to be involved in signal loss during sample screening on peptide chip, we can exclude probe proteolysis as protease inhibitors had no significant effect on peptide chip stability. Nevertheless, it is recommended to check for protease activities in samples before running large screening. The accessibility to peptides does not seem to limit the signal: steric hindrance and/or chip fouling due to partial regeneration or poor chip surface saturation would lead to an increase in base line, which is not the

The influence of peptide grafting process suggests a release of the probes during the experiments. However, in this case, the reduction of signal should be independent of the peptide, which is actually not the case. It is worth to notice that SPR signal is much lower when diazonium protocol was used. We suggest that this is due to a weaker grafting efficiency, leading to fewer immobilized peptide on the chip. Thus, SPR signal is lower and the competition for Ab binding favours high-affinity Ab. As discussed in § 3.7, high-affinity Ab keep their capacity to bind their epitopes even after epitope conformational changes, thus leading to a more stable signal. This is supported by the slope values obtained for the dissociation curves (12 ± 2.6x10-3 %reflectivity/min and 4 ± 0.9x10-3 %reflectivity/min for

Altogether, our results suggest that the main reason for the loss of SPR signal during the experiments is a change in the conformation of the grafted peptides, probably induced by the successive injection/regeneration cycles. This conformational change impairs peptide recognition by some Ab, selecting the Ab possessing a high affinity for the probe. The signal loss, due to a decrease in Ab amounts that bind to peptides, depends on both the peptide (according to its conformational stability) and the Ab (according to the targeted epitope via its accessibility upon conformational change of the peptide). Saturation process can also impact peptide stability, depending on the nature of peptidechip surface interactions, some of them being able to induce epitope masking. Involvement of peptide conformational change in signal loss is strengthened by the improvement of the signal stability observed for some peptides when glycerol was added

This study points out several ways to both reduce and correct the signal loss occurring on

ppy and diazonium respectively, first C131 injection).

peptide chip upon multiple injection/regeneration cycles.

improves the results.

in the running solutions.

**4. Conclusion** 

case.


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