Conventional wisdom suggests that widely utilized self-assembled alkylthiolate monolayers on gold are too unstable to last more than several days when exposed to complex fluids such as raw serum at body temperature. Demonstrated here is that these monolayers can not only last at least 1 week under such harsh conditions but that significant applied value can be captured for continuous electrochemical aptamer biosensors. Electrochemical aptamer biosensors provide an ideal tool to investigate monolayer degradation, as aptamer sensors require a tightly packed monolayer to preserve sensor signal vs background current and readily reveal fouling by albumin and other solutes when operating in biofluids. Week-long operation in serum at 37 °C is achieved by (1) increasing van der Waals interactions between adjacent monolayer molecules to increase the activation energy required for desorption, (2) optimizing electrochemical measurement to decrease both alkylthiolate oxidation and electric-field-induced desorption, and (3) mitigating fouling using protective zwitterionic membranes and zwitterion-based blocking layers with antifouling properties. This work further proposes origins and mechanisms of monolayer degradation in a logical stepwise manner that was previously unobservable over multiday time scales. Several of the observed results are surprising, revealing that short-term improvements to sensor longevity (i.e., hours) actually increase sensor degradation in the longer term (i.e., days). The results and underlying insights on mechanisms not only push forward fundamental understanding of stability for self-assembled monolayers but also demonstrate an important milestone for continuous electrochemical aptamer biosensors.

Conventional wisdom suggests that widely-utilized self-assembled alkylthiolate monolayers on gold are too unstable to last more than several days when exposed to complex fluids such as raw serum at body temperature. Demonstrated here is that these monolayers can not only last at least one week under such harsh conditions, but that significant applied value can be captured for continuous electrochemical aptamer biosensors. Electrochemical aptamer biosensors provide an ideal tool to investigate monolayer degradation, as aptamer sensors require a tightly-packed monolayer to preserve sensor signal versus background current and readily reveal fouling by albumin and other solutes when operating in biofluids. Week-long operation in serum at 37 ˚C is achieved by: (1) assembling monolayers on gold with roughness that promotes small alkylthiolate monolayer defect sizes; (2) increasing van der Waals interactions between adjacent monolayer molecules to increase the activation energy required for desorption; (3) optimizing electrochemical measurement to decrease both alkylthiolate oxidation and electric-field induced desorption; (4) mitigating fouling by using protective zwitterionic membranes and zwitterion-based blocking layers with antifouling properties. This work further proposes origins and mechanisms of monolayer degradation in a logical stepwise manner that was previously unobservable over multi-day time scales. Several of the observed results are surprising, revealing that short term improvements to sensor longevity (i.e., hours) actually increase sensor degradation in the longer term (i.e., days). The results and underlying insights on mechanisms not only push forward fundamental understanding of stability for self-assembled monolayers, but demonstrate an important milestone for continuous electrochemical aptamer biosensors.

Real-time continuous monitoring of proteins in-vivo holds great potential for personalized medical applications. Unfortunately, a prominent knowledge gap exists in the fundamental biology regarding protein transfer and correlation between interstitial fluid and blood. In addition, technological sensing will require affinity-based platforms that cannot be robustly protected in-vivo and will therefore be challenged in longevity, fouling, and sensitivity over multi-day to week timelines. Here we use electrochemical aptamer-based sensors as a model system to discuss further research necessary to achieve continuous protein sensing.