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Continuous biosensors provide real-time information about biochemical processes occurring in the environment of interest and are therefore highly desirable in research, diagnostics and industrial settings. Although remarkable progress has been...

Fiber optic surface plasmon resonance (FO-SPR)-based biosensors have emerged as powerful tools for biomarker detection due to their ability for real-time analysis of biomolecular interactions, cost-effectiveness, and user-friendliness. However, as (FO-)­SPR signals are determined by the mass of the target molecules, the detection of low-molecular-weight targets remains challenging and currently requires tedious labeling and preparation steps. Therefore, in this work, we established a new concept for low-molecular-weight target detection by implementing duplexed aptamers on an FO-SPR sensor. In this manner, we enabled one-step competitive detection and could achieve significant signals, independent of the weight of the target molecules, without requiring labeling or preprocessing steps. This was demonstrated for the detection of a small molecule (ATP), protein (thrombin), and ssDNA target, thereby reaching detection limits of 72 μM, 36 nM, and 30 nM respectively and proving the generalizability of the proposed bioassay. Furthermore, target detection was successfully achieved in 10-fold diluted plasma, which demonstrated the applicability of the assay in biologically relevant matrices. Altogether, the developed one-step competitive FO-SPR bioassay opens up possibilities for the detection of low-molecular-weight targets in a fast and straightforward manner.

Throughout the past decades, fiber optic surface plasmon resonance (FO-SPR)-based biosensors have proven to be powerful tools for both the characterization of biomolecular interactions and target detection. However, as FO-SPR signals are generally related to the mass that binds to the sensor surface, multistep processes and external reagents are often required to obtain significant signals for low molecular weight targets. This increases the time, cost, and complexity of the respective bioassays and hinders continuous measurements. To overcome these requirements, in this work, cis-duplexed aptamers (DAs) were implemented on FO-SPR sensors, which underwent a conformational change upon target binding. This induced a spatial redistribution of gold nanoparticles (AuNPs) upon specific target binding and resulted in an amplified and concentrationdependent signal. Importantly, the AuNPs were covalently conjugated to the sensor, so the principle does not rely on multistep processes or external reagents. To implement this concept, first, the thickness of the gold fiber coating was adapted to match the resonance conditions of the surface plasmons present on the FO-SPR sensors with those on the AuNPs. As a result, the signal obtained due to the spatial redistribution of the AuNPs was amplified by a factor of 3 compared to the most commonly used thickness. Subsequently, the cis-DAs were successfully implemented on the FO-SPR sensors, and it was demonstrated that the DA-based FO-SPR sensors could specifically and quantitatively detect an ssDNA target with a detection limit of 230 nM. Furthermore, the redistribution of the AuNPs was proven to be reversible, which is an important prerequisite for continuous measurements. Altogether, the established DA-based FO-SPR bioassay holds much promise for the detection of low molecular weight targets in the future and opens up possibilities for FO-SPRbased continuous biosensing.