Framework for Developing a Simple Method of Analyzing Environmental Bacterial Cells: Combining Single-Round DNA Aptamer Selection with Gold Nanoparticle Sensor Development for Legionella pneumophila Dete

Abstract

Monitoring pathogenic microorganisms in public and environmental waters is important for protecting ecosystems and human health; however, current detection methods are expensive, labor-intensive, and time-consuming. Although DNA aptasensors can potentially overcome these limitations, conventional aptamer selections often fail to secure high-affinity aptamer sequences, besides being complicated and lengthy. In this study, we propose a single-round polymer-enhanced capillary transient isotachophoresis/gold nanoparticle (SR-PectI/AuNP) framework for developing simple, highly sensitive DNA aptasensors. This framework comprises two key components: SR-PectI selection, which enables semicomprehensive screening of family-level high-affinity aptamer candidates for bacterial targets, and sensor development based on colorimetric changes in aptamer-conjugated AuNPs. Notably, the entire SR-PectI/AuNP framework is completed within a short duration. In this proof-of-concept study, we targeted Legionella pneumophila (Lp); seven high-affinity aptamers for Lp (1.0 ≤ Kd ≤ 14 nmol L–1; 9-fold lower than those of previously reported Lp aptamers) were successfully acquired via SR-PectI selection, followed by sensor development using AuNPs. This enabled a detection limit of 1.1 × 102 cell equivalents for Lp, lower than that for commonly used immunosensors, within 40 min. The SR-PectI/AuNP framework for environmental bacterial species provides a comprehensive approach for developing simple analytical methods for threshold-based screening.