**7. Alternative approaches to ketone sensing using Diaphorase**

The diaphorases (EC1.6.99.3) are a ubiquitous class of flavin-bound enzymes that catalyze the reduction of various dyes which act as hydrogen acceptors from the reduced form of di- and tri- phosphopyridine nucleotides, i.e. NADH, NADPH. They catalyse the following reaction:

NADPH H acceptor NADP reduced acceptor

Either NADH or NADPH may be used as reductants. However, no exchange of hydrogen between the coenzymes is catalysed. Typical acceptor molecules include dyes such as 2, 6- dichlorophenolindophenol and tetrazolium dyes and redox couples such as ferricyanide anions and ferricinium cations. The Expasy entry for diaphorase is http://enzyme.expasy.org/EC/1.6.99.3

**Figure 6.** Calibration curve (B) for the diaphorase based ketone sensor developed and manufactured in house (unpublished results). The calibration characteristics are shown over the physiologically relevant range 0 – 2.5 mmol/L -hydroxybutyrate spiked into a whole blood sample. Each concentration determination is 16 repetitions. The reaction scheme for the sensor is shown in (A). Here, the mediator is the ferricyanide anion. HBA in the blood was measured using the Randox RX Monza Chemistry Analyser, http://www.randox.com/rx%20monza.php. The test time was 7 seconds.

In this configuration, NAD+, Diaphorase, acceptor molecule and -hydroxybutyrate dehydrogenase are all formulated together in the enzyme ink and laid down on the test strip using an appropriate manufacturing method. Data generated in-house (unpublished results) using this kind of prototype test strip (with ferricyanide as the acceptor) is shown below for the clinically relevant concentration range of -hydroxybutyrate. The strip was manufactured by screen printing (*cf* above). Ferricyanide, buffer salts, NAD+, binders, diaphorase and -hydroxybutyrate dehydrogenase were mixed into a suitable enzyme ink and printed onto carbon electrodes. The operating voltage was 0.4 Volts.
