Screening Complex Biological Samples with Peptide Microarrays: the Favorable Impact of Probe Orientation via Chemoselective Immobilization Strategies on Clickable Polymeric Coatings.

The generation of robust analytical data using microarray platforms strictly relies on optimal ligand-target interaction at the sensor surface which, in turn, is inherently bound to the correct immobilization scheme of the interrogated bioprobes. In the present work, we performed a rigorous comparative analysis of the impact of peptide ligands immobilization strategy in the screening of Burkholderia cepacia complex (BCC) infections in patients affected by cystic fibrosis (CF). We generated arrays of previously validated Burkholderia-derived peptide probes, that were selectively oriented on polymeric coatings by means of different click-type reactions including thiol-maleimide, copper-catalyzed-azide-alkyne-cycloaddition (CuAAC) and strain-promoted-azide-alkyne-cycloaddition (SPAAC). We compared immobilization efficiency among the different chemoselective reactions and we evaluated diagnostic performances at a statistically significant level, also in contrast to random immobilization strategies. Our findings clearly support the favorable role of correct bioprobes orientation in discriminating seronegative from infected individuals and, in last analysis, in generating more reliable and reproducible data. Spacing biomolecules from the sensor surface by means of small hydrophilic linkers also positively affects the analytical performance and leads to increased statistical significance of data. Overall, all the click immobilization strategies that were considered displayed a good efficiency. Interestingly, SPAAC-mediated conjugation using DBCO cyclooctyne for some peptides resulted in sequence-dependent auto-fluorescence in the Cy5 emission range wavelength, which could be circumvented by using a different fluorescence detection channel. On the basis of our results, we critically discuss the immobilization parameters that need to be carefully considered for peptide ligands immobilization purposes.