Ketone β-hydroxybutyrate (HB) is an important biomarker of body's nutritional and metabolic status. For individuals with underlying metabolic conditions like diabetes, monitoring the ketone level is critical since uncontrolled ketone metabolism could lead to fatal homeostatic disorders (e.g., diabetic ketoacidosis). Wearable biosensors targeting ketone in non-invasively accessible biofluids are suitable for this application. Such biosensors can provide sample-to-answer readouts of HB with high temporal granularity and at the point-of-person, enabling timely and effective intervention. However, measuring HB in non-invasively accessible biofluids (e.g., sweat and saliva) is challenging. HB molecules become highly diluted (~10-fold) when partitioning from blood into these biofluids, with endpoint concentrations in the low micromolar range.<br/>Previously reported ketone sensors do not meet the limit of detection (LoD) requirements for non-invasive wearable monitoring, despite successfully employing dehydrogenase enzyme and electrocatalytic mediator reactions for ketone detection. These sensors are limited by their low electrocatalytic capability (stemming from the low-specific-area of the electrode) and/or use of protein-crosslinked enzyme layer with µm-thickness (limiting the analyte diffusion).<br/>Overcoming these limitations, we developed a highly-sensitive electroenzymatic sensor fabrication scheme, which exploits 1) a CNT-based mediator layer for high-performance NADH sensing as well as effective cofactor immobilization and 2) an electrodeposited enzyme layer for efficient analyte transport and reaction. The use of CNT is particularly advantageous due to its high electrocatalysis characteristics and high surface area for mediator reaction, as well as its large exposed π-electron cloud for NAD+ molecule conjugation and immobilization. By electrodepositing the dehydrogenase onto the CNT layer, the thickness of the enzyme layer can be reduced to nanometer-scale, enabling fast analyte transport without compromising the stability of the enzymatic layer. Leveraging this scheme, we achieved a NADH LoD of 0.22 µM and HB LoD of 4.4 µM. The HB sensor was also validated using spiked real biofluids (saliva: ~ 20-200 µM, within the relevant ketone concentration range of target patient groups). These results support the utility of the fabrication scheme to build the next generation of biosensors for non-invasive wearable ketone monitoring applications.

Highly-sensitive dehydrogenase-based electroenzymatic sensor fabrication scheme for non-invasive wearable ketone monitoring

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