Fabricating Reproducible, Reversible, and High Signal Change Aptasensors with Gold-Modified Nanopipettes

Aptamer-functionalized nanopipettes are an emerging class of biosensors for the label-free detection of specific molecules. While various strategies exist for immobilizing single-stranded DNA aptamers onto the inner walls of glass nanopipettes, the impact of the fabrication method on sensor sensitivity, signal change, reproducibility, and reliability remains unexplored. In this study, we compared three fabrication methods and found that sensors fabricated using gold nanoparticles (AuNPs) synthesized within the nanopipettes produced the most reproducible results while also allowing control over the modification process. In contrast, two other aptamer immobilization methods, which relied on multistep polymer coatings with aminated or thiolated aptamer coupling, were hindered by water sensitivity and uneven polymer deposition, resulting in inconsistent sensor responses. Using the AuNP-coated nanopipettes, we successfully fabricated numerous sensors of varying sizes, demonstrating that smaller nanopipettes produce greater signal changes. Sensors constructed using glass nanopipettes with diameters ranging from 22 to 30 nm exhibited large signal changes (>40%) when AuNP synthesis produced particles near the tip opening without causing blockage. However, we also observed sensors with signal changes that were significantly lower (using the same-sized glass nanopipettes), which we attributed to either minimal Au present at the tip or conversely when Au significantly blocked the probe. These results highlight the critical role of fabrication methods in maximizing the signal change, enhancing the reproducibility, and identifying how and why sensors fail. This work aims to facilitate the broader adoption of aptamer-functionalized nanopipettes in analytical sensing applications.