Solution-Phase ITC Validation of Literature-Reported Glyphosate DNA Aptamers: Affinity Ranking and an Operational Selectivity Boundary

Glyphosate is a highly polar herbicide, the reliable molecular recognition of which is complicated by co-occurring structural analogues, metabolites, and derivatives in real-world samples. Rather than reporting new aptamer discovery, this study establishes a standardized, solution-phase isothermal titration calorimetry (ITC) workflow to thermodynamically reassess two literature-reported glyphosate DNA aptamers, Seq03 and Seq05, under matched buffer composition and instrument settings. After verification of baseline stability and evaluation of heat-of-dilution contributions, ligand-to-aptamer titrations yielded apparent dissociation constants of approximately 8.14 μM for Seq03 and 40.2 μM for Seq05, enabling affinity-based prioritization of these reported candidates within the tested concentration window. To define an application-relevant selectivity boundary, we further constructed a counter-screen panel restricted to glyphosate-related chemicals, including structural analogues, metabolites, and derivatives, and evaluated all candidates using an identical ITC protocol with explicit background handling. None of the counter-screen compounds produced binding-consistent, saturable isotherms after integration and control-based interpretation; instead, their responses remained close to background heat and were therefore operationally classified as having no detectable binding under the tested conditions, including a reverse-titration format check with Glufosinate-N-acetyl. Collectively, these results position ITC as a label-free, platform-independent validation step for small-molecule aptamer benchmarking prior to analytical translation, while also highlighting that the present conclusions are bounded by the tested PBS-based conditions and the sensitivity window of the current ITC configuration.