title: Screening Complex Biological Samples with Peptide Microarrays: The Favorable Impact of Probe Orientation via Chemoselective Immobilization Strategies on Clickable Polymeric Coatings.
abstract: 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 bioprobe orientation in discriminating seronegative from infected individuals and, in the last analysis, in generating more-reliable and more-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 of 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 autofluorescence 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 ligand immobilization purposes.
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM) Via Mario Bianco, 9, 20131 Milano, Italy.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Gori
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM) Via Mario Bianco, 9, 20131 Milano, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Sola
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM) Via Mario Bianco, 9, 20131 Milano, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Gagni
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM) Via Mario Bianco, 9, 20131 Milano, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Bruni
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM) Via Mario Bianco, 9, 20131 Milano, Italy.
authors.forename: Marta
authors.initials: M
authors.lastname: Liprino
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM) Via Mario Bianco, 9, 20131 Milano, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Peri
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM) Via Mario Bianco, 9, 20131 Milano, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Colombo
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM) Via Mario Bianco, 9, 20131 Milano, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Cretich
authors.affiliation: Consiglio Nazionale delle Ricerche, Istituto di Chimica del Riconoscimento Molecolare (ICRM) Via Mario Bianco, 9, 20131 Milano, Italy.
authors.forename: Marcella
authors.initials: M
authors.lastname: Chiari
mesh_terms: D000480:Alkynes / Q000737:chemistry; D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D042602:Burkholderia cepacia complex / Q000502:physiology; D002384:Catalysis; D057930:Click Chemistry; D003300:Copper / Q000737:chemistry; D061565:Cycloaddition Reaction; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D055767:Immobilized Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011108:Polymers / Q000737:chemistry*; D040081:Protein Array Analysis; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D000480:Alkynes; D001386:Azides; D001426:Bacterial Proteins; D055767:Immobilized Proteins; D010455:Peptides; D011108:Polymers; D003300:Copper