Molecular Evolution of Specific Aptamers as Binding Molecule Equipage for the gFET-Based Quantification of the Health-Relevant Oral Space Bacterium Fusobacterium nucleatum
Description
Fusobacterium nucleatum (F. nucleatum) is an opportunistic oral pathobiont heavily implicated in the progression of severe systemic conditions, including colorectal carcinoma, neuroinflammatory disorders such as Alzheimer's disease and endometriosis. Current clinical diagnostic methodologies, such as quantitative polymerase chain reaction (qPCR) and 16S rRNA sequencing, are severely limited by high operational costs, prolonged turnaround times and the requirement for complex hardware. To address the demand for rapid, point-of-care diagnostics, we utilized a whole-bacterium Fluorescent Cell-SELEX (FluCell-SELEX) strategy, incorporating a stringent counter-selection step against the commensal Streptococcus mutans, to isolate highly specific single-stranded DNA (ssDNA) aptamers directed against native proteinaceous epitopes on the F. nucleatum cell envelope. Following next-generation sequencing and bioinformatic analysis, five elite monoclonal aptamers (FN_1 through FN_5) were identified. These candidates demonstrated robust thermodynamic binding affinities in free solution, achieving apparent dissociation constants (Kd) ranging from 0.48 nM to 7.57 nM. To validate their translational potential, these monoclonal sequences were non-covalently immobilized onto reduced graphene oxide field-effect transistor (rGO-FET) biosensors using 1-pyrenecarboxylic acid and Py-PEG linker chemistry. Empirical evaluation revealed that steric constraints on the planar surface significantly influenced binding efficacy; however, the optimized candidates (FN_1, FN_2 and FN_5) retained accessible secondary structures when tethered. The resulting rGO-FET aptasensor achieved instantaneous, label-free electronic quantification with an exceptional limit of detection of < 40 cells/mL and strict target discrimination in polymicrobial environments. This platform provides a highly sensitive, scalable foundation for personalized microbiome diagnostics and early systemic disease management.
Files
Institutions
- Universität UlmBaden-Wurttemberg, Ulm