Single-Use Electrochemical Aptamer-Based Sensors for Calibration-Free Measurements in Human Saliva via Dual-Frequency Approaches: Prospects and Challenges.

Despite the rapid growth in aptamer-based biosensor research, there remains a significant demand for aptasensors that operate without the need for sample preparation and calibration, to better facilitate real-world applications. Electrochemical aptamer-based (EAB) sensors, particularly those utilizing a dual-frequency, calibration-free approach, have shown promising advances toward commercialization. Single-use, disposable sensors represent a cost-effective solution for at-home and on-site point-of-care (POC) diagnostics. However, the development of these sensors presents unique challenges compared to in vivo monitoring and reusable platforms, with pronounced variations across sensors and batches. Motivated by these challenges, we have comprehensively investigated the dual-frequency, calibration-free approach, focusing on sensor-to-sensor and batch-to-batch variations. Our research explored the use of a nonresponsive frequency-based ratiometric method for detecting cocaine with laser-ablated, disposable EAB sensors. Additionally, to overcome the absence of nonresponsive frequencies in some aptasensors, we developed strategies to modify the aptamer structure and optimize operational conditions, effectively tailoring nonresponsive frequencies to allow for rapid result turnover. Moreover, we assessed the effects of various filter types on saliva pretreatment using liquid chromatography with tandem mass spectrometry (LCMS/MS) and developed a saliva collection workflow using an oral swab. This workflow and the disposable aptasensors developed herein achieved low μM sensitivity in saliva, with results obtainable in under 5 min, including saliva collection and processing. Furthermore, our findings indicate that certain food and drink residues in saliva can compromise sensor accuracy, highlighting an area for future refinement.