Duanghathaipornsuk Surachet, Reaver Nathan G F, Cameron Brent D, Kim Dong-Shik
Department of Chemical Engineering, 2801 W. Bancroft St., University of Toledo, Toledo, Ohio 43606, United States.
Engineering School of Sustainable Infrastructure and the Environment Environmental Engineering Sciences, University of Florida, Gainesville, Florida 32611, United States.
Langmuir. 2021 Apr 20;37(15):4647-4657. doi: 10.1021/acs.langmuir.1c00446. Epub 2021 Apr 2.
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/K) 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.
适体是与感兴趣的靶分子具有高亲和力结合的寡核苷酸。其中一个这样的靶标是糖化血红蛋白（gHb），它是用于评估血糖控制和糖尿病诊断的生物标志物。通过将适体与表面等离子体共振（SPR）传感表面偶联，可以开发一种快速、可靠且廉价的gHb检测方法。在本研究中，我们测试了由适体和防污自组装单分子层（SAMs）组成的SPR传感表面对血红蛋白（Hb）和gHb的亲和力。首先，我们通过改进的指数富集配体系统进化（SELEX）富集过程开发了一种靶向gHb的适体（GHA），并使用纳米亲和力方案测试了其对gHb的亲和力。GHA用于制备三种不同的SAM-SPR传感表面：（类型-1）GHA的单分子层直接附着在传感器表面；（类型-2）GHA附着在传感器表面的11-巯基十一烷酸（11MUA）单分子层上；（类型-3）GHA附着在传感器表面的11MUA和3,6-二氧杂-8-巯基辛-1-醇（DMOL）的二元单分子层上。通过循环伏安法和电化学阻抗谱对类型-2和类型-3表面进行表征，以确认GHA与底层单分子层结合。Hb和gHb与每个SPR传感表面相互作用的吸附动力学用于量化它们各自的亲和力。未进行防污修饰的类型-1表面的解离常数比（K/K）为9.7，而类型-3表面为809.3，这表明对于具有防污修饰的传感器表面，GHA与gHb的结合比没有修饰的表面更高。GHA对gHb增强的选择性可能归因于SAM修饰表面中包含DMOL，这减少了蛋白质非特异性吸附的干扰。结果表明，将适体与防污SAMs配对可以显著提高其对靶标的亲和力，有可能开发出新型、低成本且快速的检测方法。
