Adsorption Kinetics of Glycated Hemoglobin on Aptamer Microarrays with Antifouling Surface Modification.

Aptamers are oligonucleotides that bind with high affinity to target molecules of interest. One such target is glycated hemoglobin (gHb), a biomarker for assessing glycemic control and diabetes diagnosis. By the coupling of aptamers with surface plasmon resonance (SPR) sensing surfaces, a fast, reliable and inexpensive assay for gHb can be developed. In this study, we tested the affinity of SPR-sensing surfaces, composed of aptamers and antifouling self-assembled monolayers (SAMs), to hemoglobin (Hb) and gHb. First, we developed a gHb-targeted aptamer (GHA) through a modified Systematic Evolution of Ligands by EXponential (SELEX) enrichment process and tested its affinity to gHb using the Nano-Affi protocol. GHA was used to produce three distinct SAM-SPR-sensing surfaces: (Type-1) a SAM of GHA directly attached to a sensor surface; (Type-2) GHA attached to a SAM of 11-mercaptoundecanoic acid (11MUA) on a sensor surface; (Type-3) GHA attached to a binary SAM of 11MUA and 3,6-dioxa-8-mercaptooctan-1-ol (DMOL) on a sensor surface. Type-2 and Type-3 surfaces were characterized by cyclic voltammetry and electrochemical impedance spectroscopy to confirm that GHA bound to the underlying SAMs. The adsorption kinetics for Hb and gHb interacting with each SPR sensing surface were used to quantify their respective affinities. The Type-1 surface without antifouling modification had a dissociation constant ratio (KD,Hb/KD,gHb) of 9.7, as compared to 809.3 for the Type-3 surface, demonstrating a higher association of GHA to gHb for sensor surfaces with antifouling modifications than those without. The enhanced selectivity of GHA to gHb can likely be attributed to the inclusion of DMOL in the SAM-modified surface, which reduced interference from nonspecific adsorption of proteins. Results suggest that pairing aptamers with antifouling SAMs can significantly improve their target affinity, potentially allowing for the development of novel, low cost, and fast assays.