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

Abstract

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 (K D,Hb / K D,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.

A Review of Electrochemical Sensors for the Detection of Glycated Hemoglobin

“…Furthermore, due to a large amount of carbohydrates and protein in whole blood samples, the effective antifouling ability of the electrode can improve the affinity and specificity of detection. Duanghathaipornsuk et al [54] developed a gHb-targeted aptamer (GHA) through a modified SELEX process, and it was used to produce three distinct SAM-SPR-sensing surfaces with and without an antifouling layer. The results showed that the correlation between the HbA1c-targeted aptamer and HbA1c of the sensor surface with antifouling modification was higher than that of the sensor surface without modification, and the interference of nonspecific protein adsorption was reduced.…”

Aptamer-Based Biosensors for the Colorimetric Detection of Blood Biomarkers: Paving the Way to Clinical Laboratory Testing

“…Different research groups [ 103 , 104 , 105 ] have performed alternative selections of hemoglobin-binding DNA aptamers. However, these aptamers have been further used as biospecific elements for electrochemical, SPR, and fluorescent aptasensors with quite complicated analytical schemes and equipment [ 104 , 106 , 107 , 108 , 109 ], which can hardly be applied in routine clinical lab practice.…”

Recent Advancements in Aptamer-Based Surface Plasmon Resonance Biosensing Strategies

“…Non-specific attachment in SPR arrays has often been a critical problem that affects the reliability of assay results. To this end, Duanghathaipornsuk et al reported that the inclusion of 3,6-dioxa-8-mercaptooctan-1-ol (DMOL) in a self-assembled monolayermodified array surface can mitigate the nonspecific binding of proteins and impart more degrees of freedom to the aptamers for interacting with the targets [79]. In addition to sensing applications, SPR arrays can be used to assess the surface density of aptamer strands.…”