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

332 Dehydrogenases

blood was minimised. [15-18]

http://enzyme.expasy.org/EC/1.6.99.3

operating potential (+200 mV versus Ag/AgCl) such that the effect of electroactive agents in

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

**Figure 6.** Calibration curve (B) for the diaphorase based ketone sensor developed and manufactured in house (unpublished results).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.

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

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 protons from the substrate molecule. [19-21]

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

lose the osmotic effect of glucose quickly, but large icodextrin molecules, which are not easily transported across the peritoneal membrane, maintain an osmotic gradient. Icodextrin is not metabolised in the peritoneal cavity, but the polymer can move into the blood stream via the lymphatic system. During systemic circulation, icodextrin is mainly metabolised into maltose which accumulates due to a lack of circulating maltase. It is the accumulation in the systemic circulation of these metabolites of icodextrin that may lead to the disparity between finger stick and formal blood glucose measurement. Maltose interferes with glucose assays that use glucose dehydrogenase with cofactor pyrroloquinolinequinone (PQQ-GDH) leading to falsely increased readings.

Amperometric Glucose Sensors for Whole Blood Measurement Based on Dehydrogenase Enzymes 335

ferricyanide mediator and flavo-protein glucose dehydrogenase (FAD-GDH), are laid down on the 'bottom' palladium electrode. FAD-GDH enzyme was selected for its high substratespecificity and non-reactivity towards oxygen. The strip may be defined as a 'side-fill strip' because blood may be applied to the 400 nL sample chamber from either the left or the right side of the test strip (Figure 7). The levels of glucose in the sample are determined within 5 seconds, the BG value being shown on the meter display in geographically appropriate units

**Figure 7.** Architecture of the OneTouch Verio glucose test strip. The test strip incorporates gold and palladium electrodes that are orientated in a cofacial manner. The dimensions of the two electrodes are defined and controlled during the manufacturing process by a die-punch process. The electrodes are separated from each other by a thin plastic spacer that has a nominal thickness of 95 μm. The glucosesensitive reagents, citraconate buffer salts, potassium ferricyanide mediator, and flavoprotein glucose dehydrogenase, are laid down on the "bottom" palladium electrode. The user can apply blood to the test strip either from the right hand side or the left hand side. The glucose level is reported within 5

Manufacturing process controls and built-in signal processing compensation mechanisms eliminate the need for user calibration coding, thus reducing the potential for user error. The meter uses a novel multi-pulse signal and has an improved glucose-Hct-temperatureantioxidant compensation algorithm for higher accuracy and precision over a wide range of blood samples. The OneTouch Verio BG test strip is designed to work with meters that are technically equivalent but have different user interfaces. Currently available meters include OneTouch Verio, OneTouch VerioPro, and OneTouch VerioIQ. The performance characteristics of the OneTouch VerioPro BGMS are summarized as follows: plasma equivalent calibration; 0.4 μl sample size; FAD-GDH enzyme; 20–600 mg/dl glucose range; 20–60% Hct range; 10–90% humidity range (non-condensing); 6–44°C temperature range; and up to 3048 m altitude use. A summary of the dehydrogenase enzymes currently used in

(mg/dl or mM).

seconds of the start of the test procedure.

commercial enzyme electrodes is shown in Table 1.

Because of the oxygen insensitivity of PQQ-GDH however, there is much commercial interest in producing a mutant form of the enzyme that retains its non-reactivity to oxygen but improving its specificity with respect to D-glucose. Recently, Roche have reported the successful production of a mutant strain of PQQ-GDH which shows no cross-reactivity with maltose. [23]

Finally, it is worth mentioning that PQQ containing proteins lend themselves to an interesting electrode configuration. Whilst flavoproteins such as glucose oxidase exchange electrons with an electrode surface via small molecular weight mediators such as ferrocene, ferricyanide etc, PQQ containing enzymes can exchange electrons via cytochrome b562. This cytochrome will exchange electrons directly with the electrode surface without the need for any mediator molecule. [24] Information on PQQ-GDH can be found on the EXPASY Proteomics server at: http://expasy.org/enzyme/1.1.5.2.

A new enzyme on the market which has just recently become commercially available to the biosensor industry is the flavo-protein FAD-GDH [25] (Toyobo Develops FAD-GDH Enzyme, produced by Aspergillus for SMBG http://www.toyobo.co.jp/e/press/press31072009.htm). As its name suggests, the enzyme catalyses the oxidation of glucose but does not utilise dioxygen as a co-reactant. It can react with a number of artificial electron acceptors such as the ferricyanide anion and the ferricinium cation. The enzyme, which is isolated from *Aspergillus terreus* shows good thermal stability and high selectivity for glucose. Its absorption spectrum is typical of flavoproteins showing two distinctive peaks corresponding to the oxidised flavin cofactor at 465nm and 385 nm. In the presence of glucose the enzyme is bleached and these characteristic absorption bands disappear. [26] According to the data sheet from the manufacturer (Toyobo, Japan) the enzyme has a pH optimum of 7.0 and a KM for glucose of 67.6mM. The information on the EXPASY proteomics server relating to this enzyme is at: http://expasy.org/enzyme/1.1.99.10.

This enzyme is currently used in a number of glucose test strips including the OneTouch Verio family of test strips produced and supplied by LifeScan. The design of the OneTouch Verio BG test strip is shown in Figure 7. The test strip incorporates gold and palladium electrodes which are orientated in a co-facial manner. The dimensions of the two electrodes are defined and controlled during the manufacturing process by a die-punch process. The electrodes are separated from each other by a thin plastic spacer that has a nominal thickness of 95 μm. The glucose-sensitive reagents, citraconate buffer salts, potassium ferricyanide mediator and flavo-protein glucose dehydrogenase (FAD-GDH), are laid down on the 'bottom' palladium electrode. FAD-GDH enzyme was selected for its high substratespecificity and non-reactivity towards oxygen. The strip may be defined as a 'side-fill strip' because blood may be applied to the 400 nL sample chamber from either the left or the right side of the test strip (Figure 7). The levels of glucose in the sample are determined within 5 seconds, the BG value being shown on the meter display in geographically appropriate units (mg/dl or mM).

334 Dehydrogenases

maltose. [23]

lose the osmotic effect of glucose quickly, but large icodextrin molecules, which are not easily transported across the peritoneal membrane, maintain an osmotic gradient. Icodextrin is not metabolised in the peritoneal cavity, but the polymer can move into the blood stream via the lymphatic system. During systemic circulation, icodextrin is mainly metabolised into maltose which accumulates due to a lack of circulating maltase. It is the accumulation in the systemic circulation of these metabolites of icodextrin that may lead to the disparity between finger stick and formal blood glucose measurement. Maltose interferes with glucose assays that use glucose dehydrogenase with cofactor pyrroloquinolinequinone

Because of the oxygen insensitivity of PQQ-GDH however, there is much commercial interest in producing a mutant form of the enzyme that retains its non-reactivity to oxygen but improving its specificity with respect to D-glucose. Recently, Roche have reported the successful production of a mutant strain of PQQ-GDH which shows no cross-reactivity with

Finally, it is worth mentioning that PQQ containing proteins lend themselves to an interesting electrode configuration. Whilst flavoproteins such as glucose oxidase exchange electrons with an electrode surface via small molecular weight mediators such as ferrocene, ferricyanide etc, PQQ containing enzymes can exchange electrons via cytochrome b562. This cytochrome will exchange electrons directly with the electrode surface without the need for any mediator molecule. [24] Information on PQQ-GDH can be found on the EXPASY

A new enzyme on the market which has just recently become commercially available to the biosensor industry is the flavo-protein FAD-GDH [25] (Toyobo Develops FAD-GDH Enzyme, produced by Aspergillus for SMBG http://www.toyobo.co.jp/e/press/press31072009.htm). As its name suggests, the enzyme catalyses the oxidation of glucose but does not utilise dioxygen as a co-reactant. It can react with a number of artificial electron acceptors such as the ferricyanide anion and the ferricinium cation. The enzyme, which is isolated from *Aspergillus terreus* shows good thermal stability and high selectivity for glucose. Its absorption spectrum is typical of flavoproteins showing two distinctive peaks corresponding to the oxidised flavin cofactor at 465nm and 385 nm. In the presence of glucose the enzyme is bleached and these characteristic absorption bands disappear. [26] According to the data sheet from the manufacturer (Toyobo, Japan) the enzyme has a pH optimum of 7.0 and a KM for glucose of 67.6mM. The information on the EXPASY proteomics server relating to this

This enzyme is currently used in a number of glucose test strips including the OneTouch Verio family of test strips produced and supplied by LifeScan. The design of the OneTouch Verio BG test strip is shown in Figure 7. The test strip incorporates gold and palladium electrodes which are orientated in a co-facial manner. The dimensions of the two electrodes are defined and controlled during the manufacturing process by a die-punch process. The electrodes are separated from each other by a thin plastic spacer that has a nominal thickness of 95 μm. The glucose-sensitive reagents, citraconate buffer salts, potassium

(PQQ-GDH) leading to falsely increased readings.

Proteomics server at: http://expasy.org/enzyme/1.1.5.2.

enzyme is at: http://expasy.org/enzyme/1.1.99.10.

**Figure 7.** Architecture of the OneTouch Verio glucose test strip. The test strip incorporates gold and palladium electrodes that are orientated in a cofacial manner. The dimensions of the two electrodes are defined and controlled during the manufacturing process by a die-punch process. The electrodes are separated from each other by a thin plastic spacer that has a nominal thickness of 95 μm. The glucosesensitive reagents, citraconate buffer salts, potassium ferricyanide mediator, and flavoprotein glucose dehydrogenase, are laid down on the "bottom" palladium electrode. The user can apply blood to the test strip either from the right hand side or the left hand side. The glucose level is reported within 5 seconds of the start of the test procedure.

Manufacturing process controls and built-in signal processing compensation mechanisms eliminate the need for user calibration coding, thus reducing the potential for user error. The meter uses a novel multi-pulse signal and has an improved glucose-Hct-temperatureantioxidant compensation algorithm for higher accuracy and precision over a wide range of blood samples. The OneTouch Verio BG test strip is designed to work with meters that are technically equivalent but have different user interfaces. Currently available meters include OneTouch Verio, OneTouch VerioPro, and OneTouch VerioIQ. The performance characteristics of the OneTouch VerioPro BGMS are summarized as follows: plasma equivalent calibration; 0.4 μl sample size; FAD-GDH enzyme; 20–600 mg/dl glucose range; 20–60% Hct range; 10–90% humidity range (non-condensing); 6–44°C temperature range; and up to 3048 m altitude use. A summary of the dehydrogenase enzymes currently used in commercial enzyme electrodes is shown in Table 1.


Amperometric Glucose Sensors for Whole Blood Measurement Based on Dehydrogenase Enzymes 337

blood glucose control. [27-29] Effective glycaemic control requires frequent measurements of blood glucose in order to take necessary therapeutic interventions. Such an approach is exemplified by the use of so-called 'continuous glucose monitoring' (CGM) apparatus and methodologies that are used by a growing number of patients to monitor their diabetes condition. Such systems are composed of a probe that is inserted into the body such that it contacts glucose containing liquids in the body, such as interstitial fluid. These CGM systems are designed to operate over extended periods of time, typically over a number of days or longer. In reality CGM is a misnomer, inasmuch as the device samples in an episodic manner, but on a sufficiently high frequency to distinguish such devices from single measurement episodic systems. Nevertheless, compared with episodic selfmonitoring blood glucose (SMBG), CGM follows blood glucose dynamics and hence, provides patients and healthcare professionals with not only current blood glucose levels, but also real-time rate and direction of changes. Blood glucose thresholds can be set to alert for possible dangerous trends, for instance rapid blood glucose descents that may lead to hypoglycemia. Advances in CGM research and development are also critical to realize

"artificial pancreas" of a closed-loop system in conjunction with an insulin pump.

inorganic layers,[51,52] and blend in carbon pastes. [53]

Enzyme catalysed electrochemical biosensors have been the most successful technology for the commercialized SMBG products (as discussed in the preceding part of this chapter). For CGM, enzymes are also employed as the means of target analyte recognition, coupled to electrochemical transduction methods for determination of the analyte of interest. Correspondingly, such systems have been so far limited to the use of a redox enzyme, such as glucose oxidase (GOx) in which the prosthetic group is intimately associated with the enzyme, such that it cannot diffuse or leach away over the duration of sensor operation.[30] Recently commercialized flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH, EC 1.1.99.10), where the FAD moiety forms an integral part of the enzyme, has attracted great attention for blood glucose monitoring because of its advantages over GOx in terms of insensitivity to molecular oxygen. However, like many redox enzymes, direct electron transfer between FAD-GDH and electrode surface cannot occur because the active centre FAD is insulated by the large proteins. Therefore, mediators are usually employed to shuttle electron between the FAD moiety and electrode surface. An example of such mediators is potassium ferricyanide used for LifeScan OneTouch Verio family of SMBG products. However, use of small molecular mediators in biosensors faces tremendous technical challenges for CGM which requires essentially all the reactive reagents immobilized without leaching out from the electrodes to achieve long-term measurement stability and to meet biocompatibility requirements, in particular for in-vivo applications. An attractive approach to tackle this challenge is to use a polymeric mediator which has mediator moieties chemically attached to polymer chains. Because of its large molecular size, the polymeric mediator can be co-immobilized with enzyme at electrode by various means, including surface grafting, [31-34] layer-by-layer surface adsorption, [35-37] retention behind semi-permeable dialysis membranes,[38-40] physical entrapment [41-43] or cross-link in hydrogels, [44-47] entrapment in electropolymerized [48,49] or chemically formed layers [50] or in

**Table 1.** Commonly used dehydrogenase enzymes for Commercial Self testing Biosensors

Shown below are examples of commercially available test strips for home use which incorporate dehydrogenase based enzymes.


(Photograph courtesy of Mr Christopher Leach, LifeScan Scotland Ltd., Inverness, UK).

**Figure 8.** Examples of commercially available test strips for self-testing utilising dehydrogenases.
