biostudies-literatureHinson CMNICHD NIH HHSNIDDK NIH HHSFoundation for the National Institutes of HealthWelch FoundationWelch Regents ChairNIGMS NIH HHSNational Science Foundation14856-14865https://www.ebi.ac.uk/biostudies/studies/S-EPMC8982273biostudies-literatureUnknown37(51)Silica passivating agents have shown great success in minimizing nonspecific protein binding to glass surfaces for imaging and microscopy applications. Amine-derivatized surfaces are commonly used in conjugation with amide coupling agents to immobilize peptides/proteins through C-terminal or side-chain carboxylic acids. In the case of the single-molecule fluorosequencing of peptides, attachment occurs via the C-terminus and nonspecific surface binding has previously been a source of error in peptide identification. Here, we employ fluorosequencing as a high-throughput, single-molecule sensitivity assay to identify and quantify the extent of nonspecific binding of peptides to amine-derivatized surfaces. We show that there is little improvement when using common passivating agents in combinaLangmuir : the ACS journal of surfaces and colloidsStudies of Surface Preparation for the Fluorosequencing of Peptides.PMC8982273R01 DK110520F-0046F-1515R35 GM122480STTR Grant #1938726R01 HD085901Hinson CMSwaminathan JAnslyn EVShannon CERivera SBardo AMMarcotte EMfalseStudies of Surface Preparation for the Fluorosequencing of Peptides.Silica passivating agents have shown great success in minimizing nonspecific protein binding to glass surfaces for imaging and microscopy applications. Amine-derivatized surfaces are commonly used in conjugation with amide coupling agents to immobilize peptides/proteins through C-terminal or side-chain carboxylic acids. In the case of the single-molecule fluorosequencing of peptides, attachment occurs via the C-terminus and nonspecific surface binding has previously been a source of error in peptide identification. Here, we employ fluorosequencing as a high-throughput, single-molecule sensitivity assay to identify and quantify the extent of nonspecific binding of peptides to amine-derivatized surfaces. We show that there is little improvement when using common passivating agents in combina2021-01-01T00:00:00Z2021 Dec2026-05-09T16:28:41.386Z2025-04-05T22:18:29.48ZS-EPMC89822733490483310.1021/acs.langmuir.1c02644