Nalaoh Phattananawee, Clark Vincent, Arroyo-Currás Netzahualcóyotl, Jenkins David M
Department of Chemistry, University of Tennessee Knoxville, Knoxville, Tennessee 37996, United States.
Chemistry-Biology Interface Program, Johns Hopkins University, Baltimore, Maryland 21218, United States.
ACS Sens. 2025 Aug 22;10(8):6039-6047. doi: 10.1021/acssensors.5c01548. Epub 2025 Aug 7.
Nucleic acid-based sensors (NBEs) are used for biomolecular detection and can enable continuous, real-time molecular monitoring . NBEs are typically constructed via thiol self-assembled monolayers (SAMs) on gold electrodes, consisting of redox-reporter-modified oligonucleotides diluted within an alkylthiol monolayer. However, the limited stability of thiol SAMs when chronically exposed to biological fluids restricts the long-term lifespan of NBEs. In contrast, N-heterocyclic carbenes (NHCs) are monolayer-forming ligands that offer improved surface stability versus thiols for biological applications, but to date, the synthetic routes to attach biomolecules to NHC surfaces rely solely on azides tethered to the NHC. As an alternative, employing strain-promoted azide-alkyne cycloadditions (SPAAC) type building blocks on the NHCs, including bicyclo[6.1.0]nonyne (BCN) and dibenzoazacyclooctyne (DBCO), allows for a broader array of azide-modified biomolecules to be employed. In this study, three new masked NHCs for the SPAAC Click reactions were synthesized and characterized. These SPAAC-modified NHCs successfully clicked and transformed their cyclooctynes into triazoles with organic azides at room temperature. The best NHC of the three was deposited onto gold electrode surfaces and successfully achieved coupling of redox-reporter-modified aptamer oligonucleotides, as verified via the electrochemical reduction of their reporter, methylene blue. While these results confirm the success of using cyclooctyne-based NHCs and azide-modified aptamers for copper-free SPAAC Click reactions, more research is needed to improve NHC packing to prevent the generation of hydrogen peroxide on gold during electrochemical interrogation of NBEs. Nevertheless, this study opens novel opportunities for surface modification with an expansive array of aptamers and, potentially, other biomolecules for electrochemical sensing applications.
基于核酸的传感器（NBEs）用于生物分子检测，并能够实现连续、实时的分子监测。NBEs通常通过金电极上的硫醇自组装单分子层（SAMs）构建，由在烷基硫醇单分子层中稀释的氧化还原报告基团修饰的寡核苷酸组成。然而，硫醇SAMs在长期暴露于生物流体时稳定性有限，限制了NBEs的长期使用寿命。相比之下，N-杂环卡宾（NHCs）是形成单分子层的配体，在生物应用中比硫醇具有更高的表面稳定性，但迄今为止，将生物分子连接到NHC表面的合成路线仅依赖于连接到NHC的叠氮化物。作为一种替代方法，在NHCs上使用应变促进的叠氮化物-炔烃环加成（SPAAC）类型的构建块，包括双环[6.1.0]壬炔（BCN）和二苯并氮杂环辛炔（DBCO），可以使用更广泛的叠氮化物修饰的生物分子。在本研究中，合成并表征了三种用于SPAAC点击反应的新型掩蔽NHCs。这些经SPAAC修饰的NHCs在室温下成功地与有机叠氮化物发生点击反应，并将其环辛炔转化为三唑。这三种NHCs中性能最佳的被沉积在金电极表面，并成功实现了氧化还原报告基团修饰的适体寡核苷酸的偶联，这通过其报告基团亚甲基蓝的电化学还原得到验证。虽然这些结果证实了使用基于环辛炔的NHCs和叠氮化物修饰的适体进行无铜SPAAC点击反应的成功，但仍需要更多研究来改善NHC的堆积，以防止在对NBEs进行电化学检测时在金表面产生过氧化氢。尽管如此，本研究为使用大量适体以及潜在地使用其他生物分子进行表面修饰以用于电化学传感应用开辟了新的机会。
