**6. Ketone sensors which incorporate a NAD-linked dehydrogenase**

In addition to glucose, another important analyte which is of particular relevance to diabetic patients is the ketone body, -hydroxybutyrate. Diabetic ketoacidosis (DKA), a condition were the level of ketone bodies in the blood are elevated, is a problem that occurs in people with diabetes. It occurs when the body cannot use sugar (glucose) as a fuel source because there is no insulin or not enough insulin. Fat is used for fuel instead. People with type 1 diabetes do not have enough insulin, a hormone the body uses to break down sugar (glucose) in the blood for energy. When glucose is not available, fat is broken down instead.

As fats are broken down, acids called ketones build up in the blood and urine. In high levels, ketones are poisonous. This condition is known as ketoacidosis. Blood glucose levels rise (usually higher than 300 mg/dL) because the liver makes glucose to try to combat the problem. However the cells cannot pull in that glucose without insulin. Diabetic ketoacidosis is often the first sign of type 1 diabetes in people who do not yet have other symptoms. It can also occur in someone who has already been diagnosed with type 1 diabetes. Infection, injury, a serious illness, or surgery can lead to diabetic ketoacidosis in people with type 1 diabetes. Missing doses of insulin can also lead to ketoacidosis in people with diabetes. People with type 2 diabetes can develop ketoacidosis, but it is rare. It is usually triggered by a severe illness. In ketoacidosis, the body fails to adequately regulate ketone production causing such a severe accumulation of keto acids that the pH of the blood is substantially decreased. In extreme cases ketoacidosis can be fatal. 14 Despite considerable advances in diabetes therapy, key epidemiological figures related to DKA remained nearly unchanged during the last decades at a global level. Prevention of DKA – especially in sick day management – relies on intensive self-monitoring of blood glucose and subsequent, appropriate therapy adjustments. Self-monitoring of ketone bodies during hyperglycemia can provide important, complementary information on the metabolic state. Both methods for self-monitoring of ketone bodies at home are clinically reliable and there is no published evidence favouring one method with respect to DKA prevention.

330 Dehydrogenases

carried out at a separate printer/dryer station.

and inks that are used to define structural components of the device (insulating inks to define the electrode areas and geometries, adhesive inks to provide three dimensional elements such as capillary spacers and coloured inks to provide branding and product identification.) Each of these individual layers normally requires a separate screen and is

The blood monitors (the systems that the user receives) are made up of three main parts: the Optium meter, the Optium Plus blood glucose electrodes, and the Optium blood ß-Ketone electrodes. When the blood sample is applied to the electrode, the glucose or ketone (ßhydroxybutyrate) in the blood reacts with the chemicals on the electrode. This reaction produces a small electrical current that is measured and the result is displayed by the sensor. Optium Xceed monitors are designed for testing blood obtained from a finger prick, but you can also use it to test blood from other, less painful, sites such as the base of the thumb, forearm or upper arm. A recent study published in *Clinical Chemistry and Laboratory Medicine* [13] concluded that the Optium Blood glucose test strip had a within-run imprecision coefficient of variation (CV) of 4.2%. Good response linearity was found in glucose concentrations in the range 31–444 mg/dL (1.7–24.7 mmol/L). In the concentration range studied, the glucose meter error was 5.14% and the linear regression equation was y = 0.91x +6.2 (r=0.984) against a Modular P clinical analyzer. The Passing-Bablok agreement test indicated good concordance of results. However, for glucose concentrations <100 mg/dL (5.6 mmol/L) (n=69) the error was 6.82% with regression equation y=0.86x+5.9 (r=0.757). Between-lot differences amounted to 0.7%–18.2%. The authors concluded that meter had good precision and accuracy when compared to the laboratory method and met the quality recommendations of the National Committee of Clinical Laboratory Standards (NCCLS, currently the Clinical Laboratory Standards Institute), the National Academy of Clinical

Biochemistry (NACB) and the International Organization for Standardization (ISO).

**6. Ketone sensors which incorporate a NAD-linked dehydrogenase** 

In addition to glucose, another important analyte which is of particular relevance to diabetic patients is the ketone body, -hydroxybutyrate. Diabetic ketoacidosis (DKA), a condition were the level of ketone bodies in the blood are elevated, is a problem that occurs in people with diabetes. It occurs when the body cannot use sugar (glucose) as a fuel source because there is no insulin or not enough insulin. Fat is used for fuel instead. People with type 1 diabetes do not have enough insulin, a hormone the body uses to break down sugar (glucose) in the blood for energy. When glucose is not available, fat is broken down instead. As fats are broken down, acids called ketones build up in the blood and urine. In high levels, ketones are poisonous. This condition is known as ketoacidosis. Blood glucose levels rise (usually higher than 300 mg/dL) because the liver makes glucose to try to combat the problem. However the cells cannot pull in that glucose without insulin. Diabetic ketoacidosis is often the first sign of type 1 diabetes in people who do not yet have other symptoms. It can also occur in someone who has already been diagnosed with type 1 diabetes. Infection, injury, a serious illness, or surgery can lead to diabetic ketoacidosis in people with type 1 Ketone sensitive test strips can be manufactured using NAD-linked enzyme, -hydroxybutyrate dehydrogenase and chemically modified electrodes like the ones described above. It is interesting to note however that in the case of this particular enzyme representatives of the common classes of quinoid NADH redox mediator, including Meldola Blue, 4-methyl-1,2 benzoquinone, 1-methoxy phenazine methosulphate and 2,6-dichloroindophenol, were shown to inhibit the NAD-dependent enzyme -hydroxybutyrate dehydrogenase, severely limiting their utility in the construction of a stable biosensor electrode for the ketone body hydroxybutyrate. [14] The authors speculated that this was due to 1,4-nucleophilic addition with enzyme amino acid residues such as cystine present on the enzyme. Consequently, this mode of inhibition is overcome through the use of mediators such as 1, 10-phenanthroline quinone (Figure 5.)

**Figure 5.** Chemical structure of 1,10 Phenanthroline quinone (oxidised form). The oxidation potential of this molecule is 0.1 Volt vs Ag/AgCl. The electrochemical reduction of 1,10-phenanthroline-5,6-quinone, like other quinones, is reversible and occurs by 2e- transfer in a single step in aqueous solution and by two 1 e-transfer steps in aprotic media.

This technology resulted in the launch of the MediSense® Optium™ β-Ketone electrode. The strip was stable, (≤10% loss in response at 30 C versus 4 C) with a long shelf life of 18 months. Diabetics were able to determine their -hydroxybutyrate level with good precision (0.43 mM 3-OHB, 10.5% CV; 1.08 mM, 5.9%; 3.55 mM, 3.2%; n = 20 per level) and accuracy (versus reference assay: slope = 0.98; intercept = 0.02 mM, r = 0.97, n = 120) over the range 0.0–6.0 mM in 30 s using a small volume of blood (5 μl). The electrode had a low operating potential (+200 mV versus Ag/AgCl) such that the effect of electroactive agents in blood was minimised. [15-18]

Amperometric Glucose Sensors for Whole Blood Measurement Based on Dehydrogenase Enzymes 333

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.

**dehydrogenase** 

protons from the substrate molecule. [19-21]

**8. Glucose test strips using PQQ linked and FAD-linked glucose** 

PQQ-GDH (pyrrolo quinoline quinone glucose dehydrogenase) belongs to a class of enzymes called quinoproteins which require ortho-quinone cofactors to oxidize a wide variety of alcohols and amines to their corresponding aldehydes and ketones. The soluble quinoprotein Glucose Dehydrogenase, EC 1.1.99.17, (sGDH), systematic name D-glucose: (pyrrolo-quinoline-quinone)1-oxidoreductase, uses pyrrolo-quinoline quinone (PQQ) as a cofactor. sGDH is a strongly basic (pI = 9.5) dimeric enzyme of identical subunits. One monomer (50 kDa, 454 residues) has been reported to bind one PQQ molecule and two Ca(II) ions. One of the Ca(II) ions is required for activation of the cofactor; the other is needed for functional dimerisation of the protein. sGDH oxidizes a wide range of mono-and disaccharides and is able to donate electrons to several neutral or cationic artificial electron acceptors, including short artificial ubiquinone homologues. The natural electron acceptor of PQQ-GDH is ubiquinone although the enzyme will react with a variety of artificial acceptors such as the ferricyanide anion ion and the ferricinum cation. The oxidised form of PQQ can be converted into the reduced form PQQH2 by the transfer of 2 electrons and two

There are two types of PQQ-GDH enzymes that can be considered for biosensor design. One is intracellular and soluble (sPQQ-GDH) whereas the other molecule is insoluble and firmly bound to the outer surface of the cytoplasmic membrane (mPQQ-GDH). mPQQ-GDH is very selective for glucose but has the disadvantage that it requires extensive solubilisation and stabilisation with detergent molecules[22] . For these reasons, mPQQ-GDH has not been successfully commercialised for biosensor application. It can oxidise a number of monosaccharides, in addition to glucose, such as maltose, mannose and lactose. Consequently, patients that have high levels of, for example maltose in the blood (which could result as a side effect of peritoneal dialysis) or have an inbred genetic disorder resulting in impaired carbohydrate metabolism, would obtain an inaccurate high reading when testing with glucose electrodes incorporating this enzyme. The increasing demand for dialysis and slower growth in capacity for haemodialysis has reinforced the need for an integrated approach to providing dialysis. Peritoneal dialysis is the preferred option for a proportion of patients with end stage renal failure. Peritoneal dialysis fluid usually contains glucose as an osmotic agent to enable water to pass across the peritoneum. Some patients
