Continuous in vivo monitoring of small molecule biomarkers requires biosensors with reversibility, sensitivity in physiologically relevant ranges, and biological stability. Leveraging the real-time, label-free detection capability of surface plasmon resonance (SPR) technology, a molecularly responsive hydrogel film is introduced to enhance small molecule sensitivity. This advanced biosensing platform utilizes split-aptamer-cross-linked hydrogels (aptagels) engineered using 8-arm poly(ethylene glycol) macromers, capable of directly and reversibly detecting vancomycin. Investigation through SPR and optical waveguide mode, along with quartz crystal microbalance with dissipation (QCM-D) monitoring, reveals that the reversible formation of analyte-induced ternary molecular complexes leads to aptagel contraction and significant refractive index changes. Optimization of aptamer cross-link distribution and complementarity of split-aptamer pairs maximizes conformational changes of the aptagel, demonstrating a detection limit of 160-250 nM for vancomycin (6-9 fold improvement over monolayer counterpart) with a broad linear sensing range up to 1 mM. The aptagel maintains stability over 24 h in blood serum and 5 weeks in diluted blood plasma (mimicking interstitial fluid). This structurally responsive aptagel platform with superior stability and sensitivity offers promising avenues for continuous in vivo monitoring of small molecules.

BioMed 10 Institute Heidelberg 69120 Germany

Central Nervous System Diseases Research Boehringer Ingelheim Pharma GmbH and Co KG Biberach an der Riß 88400 Germany

Department of Biophysics Chemical and Macromolecular Physics Faculty of Mathematics and Physics Charles University Prague 150 06 Czech Republic

Faculty of Biotechnology Mannheim University of Applied Sciences Mannheim 68163 Germany

FZU Institute of Physics Czech Academy of Sciences Prague 180 00 Czech Republic

LiST Life Sciences Technology Danube Private University Wiener Neustadt 2700 Austria

Macromolecular Chemistry Department of Chemistry and Biology University of Siegen Siegen 57076 Germany

School of Biological and Health Systems Engineering Arizona State University Tempe Arizona 85281 United States

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