Disposable screen-printed electrochemical sensing strips for rapid decentralized measurements of salivary ketone bodies: Towards therapeutic and wellness applications

The interest in ketone bodies (KBs) has intensified recently as they play significant roles in healthcare, nutrition, and wellness applications. We present a disposable electrochemical sensing strip for rapid decentralized detection of β-hydroxybutyrate (HB), one of the dominant physiological KBs, in saliva. The new salivary enzymatic HB sensor strip relies on a gold-coated screen-printed carbon electrode modified with a reagent layer containing toluidine blue O (TBO mediator), β-hydroxybutyrate dehydrogenase (HBD enzyme), and the HBD cofactor nicotinamide adenine dinucleotide (NAD+ coenzyme), along with carbon nanotubes (CNTs) and chitosan (Chit) for enhancing the sensor's sensitivity and for encapsulating the enzyme and its cofactor, respectively. The systematic optimization resulted in an attractive analytical performance, with a rapid response time within 60 s, a wide HB dynamic detection range from 0.1 to 3.0 mM along with a low limit of detection (50 μM HB) in an artificial saliva medium. The strip displays high selectivity for HB over acetoacetate (AcAc) and other interferences (i.e., acetaminophen, ascorbic acid, glucose, lactic acid, and uric acid), good reproducibility, and high stability towards temperature or pH effects. The new disposable sensing strip system, coupled with a hand-held electrochemical analyzer, showed rapid HB monitoring in human saliva samples collected from healthy volunteers, with similar temporal profiles to those obtained in parallel capillary blood measurements in response to the intake of keto supplements. This strip enables efficient, reliable, and near real-time salivary HB detection to track non-invasively the dynamics of HB concentrations after intaking commercial supplements towards diverse healthcare and nutrition applications.