**1. Introduction**

318 Dehydrogenases

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Self-monitoring blood glucose (SMBG) is an important component of modern therapy for diabetes mellitus. SMBG has been recommended for people with diabetes and their health care professionals in order to achieve a specific level of glycaemic control and to prevent hypoglycaemia. The goal of SMBG is to collect detailed information about blood glucose levels at many time points to enable maintenance of a more constant glucose level by more precise regimens. It can be used to aid in the adjustment of a therapeutic regimen in response to blood glucose values and to help individuals adjust their dietary intake, physical activity, and insulin doses to improve glycaemic control on a day-to-day basis.

SMBG can aid in diabetes control by: (http://www.diabetes.co.uk/blood-glucose/bloodglucose-self-monitoring.html)

© 2012 Cardosi and Liu, licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

condition that diabetics try to avoid through proper action. To this end, regular testing can predict dangerous drop in blood glucose concentration which can lead to hypoglycaemia.

Amperometric Glucose Sensors for Whole Blood Measurement Based on Dehydrogenase Enzymes 321

within the target range, infrequent SMBG monitoring is appropriate. In such cases, patients can use SMBG data as biofeedback at times of increased stress or changes in diet or physical

It is important to point out however that this debate is not straightforward, however. One factor that is often overlooked is that the numbers obtained by testing are only one part of the picture, which requires additional data to be complete. For example, it is important to relate the number to what and when the patient last ate. The patient's exercise regime must also be considered, as well as when and how much medication has been taken. If this were not complicated enough, the patient (and physician) need to consider factors such as whether the patient has recently been ill, or even subjected to high levels of stress, which can

Blood glucose meters which utilise an enzyme electrode (a term first coined by Updike and Hicks in the late sixties [6]) as the glucose sensing element are particularly suitable medical

All the commercial electrochemical meter systems typically comprise two components. The meter - applies potential differences in a programmed sequence to the sensor, collects current data and analyses the current time response of the sensor, records and displays results. The enzyme electrode (or biosensor) test strip - collects the blood sample, the sample undergoes an enzymatic chemical reaction followed by an electrochemical detection step. The patient simply inserts the enzyme electrode into the meter and applies a small drop of blood to the sensor. After a short delay (typically 5 seconds) the blood glucose value is

The advantages offered by biosensors in SMBG arise for the following reasons. Blood is a complex fluid and glucose levels vary widely over time in a single patient, many factors besides glucose vary in blood from healthy, patients (haematocrit, oxygen levels, and metabolic by-products) therefore great specificity is a prime requirement. In addition, patients with diabetes may have a wide range of other medical problems creating even greater variation in their blood. Finally, biosensors can be used directly in the blood without requiring major modifications to the biological sample (increased temperature or pressure,

The enzyme electrodes commonly used in SMBG can be defined as a combination of any electrochemical probe with a thin layer of enzyme based reagent that is selective for glucose (*or other important analytes such as β-hydroxybutyrate*). In these devices, the function of the enzyme is to provide selectivity by virtue of its biological affinity for a particular substrate molecule. For example, an enzyme is capable of catalysing a particular reaction of a given substrate even though other isomers of that substrate or similar substrates may be present.

Typically, the progress of the enzyme reaction (which is related to the concentration of analyte) is monitored by the rate of formation of product or the disappearance of a reactant. If either the product or reactant is electroactive, then the progress of the reaction can be monitored directly using amperometry. In this technique, current flow is measured in

activity. [4, 5]

distort the picture.

devices for SMBG.

displayed by the meter in mg/dL or mmol/L.

dramatic pH changes, addition of highly reactive chemicals, etc).

 is important for undertaking dangerous tasks which could be influenced by high or low blood sugar, such as driving and handling dangerous machinery

Disadvantages are mainly seen when either the patient lacks motivation to test or does not have sufficient education on how to interpret the results to make sufficient use of home testing equipment. Where this is the case, the following disadvantages may outweigh the potential benefits:

Numerous trials have been carried out to determine the true impact of SMBG on glycaemic control. Some, including randomized, controlled trials, have demonstrated the efficacy of SMBG. Among patients with type 1 diabetes, SMBG has been associated with improved health outcomes. [1] Specifically, increasing frequency of SMBG was linearly correlated with reductions in HbA1c among type 1 patients in Scotland. [2] Among patients with type 2 diabetes, a higher frequency of SMBG was associated with better glycaemic control among insulin-treated patients who were able to adjust their regimen. [3]

SMBG works by having patients perform a number of glucose tests each day or each week. The test most commonly involves pricking a finger with a lancet device to obtain a small blood sample, applying a drop of blood onto a reagent strip (typically an enzyme electrode), and determining the glucose concentration by inserting the strip into an electronic meter for an automated reading. Test results (a measure of the glucose concentration in the blood sample) are then recorded either in a logbook or stored in the glucose meter's electronic memory. People with diabetes can be taught to use their SMBG results to correct any deviations out of a desired target range by changing their carbohydrate intake, exercising, or using more or less insulin.

The frequency with which patients with diabetes should monitor their blood glucose level varies from person to person. Most experts agree that insulin-treated patients should monitor blood glucose at least four times a day, most commonly fasting, before meals, and before bed. In addition, patients using insulin can benefit by obtaining postprandial blood glucose readings to help them more accurately adjust their insulin regimen. A positive correlation between frequency of SMBG and glycaemic control among patients with insulin-treated type 1 or type 2 diabetes has been demonstrated. [1-3] Patients treated with intermediate, shortacting, or rapid-acting insulin may benefit from SMBG data to make adjustments in their regimen.

For patients with type 2 diabetes, optimal SMBG frequency varies depending on the pharmaceutical regimen and whether patients are in an adjustment phase or at their target for glycaemic control. If a patient is on a stable oral regimen with HbA1c concentration within the target range, infrequent SMBG monitoring is appropriate. In such cases, patients can use SMBG data as biofeedback at times of increased stress or changes in diet or physical activity. [4, 5]

320 Dehydrogenases

hypoglycaemia.

potential benefits:

using more or less insulin.

regimen.

condition that diabetics try to avoid through proper action. To this end, regular testing can predict dangerous drop in blood glucose concentration which can lead to

is important for undertaking dangerous tasks which could be influenced by high or low

Disadvantages are mainly seen when either the patient lacks motivation to test or does not have sufficient education on how to interpret the results to make sufficient use of home testing equipment. Where this is the case, the following disadvantages may outweigh the

Numerous trials have been carried out to determine the true impact of SMBG on glycaemic control. Some, including randomized, controlled trials, have demonstrated the efficacy of SMBG. Among patients with type 1 diabetes, SMBG has been associated with improved health outcomes. [1] Specifically, increasing frequency of SMBG was linearly correlated with reductions in HbA1c among type 1 patients in Scotland. [2] Among patients with type 2 diabetes, a higher frequency of SMBG was associated with better glycaemic control among

SMBG works by having patients perform a number of glucose tests each day or each week. The test most commonly involves pricking a finger with a lancet device to obtain a small blood sample, applying a drop of blood onto a reagent strip (typically an enzyme electrode), and determining the glucose concentration by inserting the strip into an electronic meter for an automated reading. Test results (a measure of the glucose concentration in the blood sample) are then recorded either in a logbook or stored in the glucose meter's electronic memory. People with diabetes can be taught to use their SMBG results to correct any deviations out of a desired target range by changing their carbohydrate intake, exercising, or

The frequency with which patients with diabetes should monitor their blood glucose level varies from person to person. Most experts agree that insulin-treated patients should monitor blood glucose at least four times a day, most commonly fasting, before meals, and before bed. In addition, patients using insulin can benefit by obtaining postprandial blood glucose readings to help them more accurately adjust their insulin regimen. A positive correlation between frequency of SMBG and glycaemic control among patients with insulin-treated type 1 or type 2 diabetes has been demonstrated. [1-3] Patients treated with intermediate, shortacting, or rapid-acting insulin may benefit from SMBG data to make adjustments in their

For patients with type 2 diabetes, optimal SMBG frequency varies depending on the pharmaceutical regimen and whether patients are in an adjustment phase or at their target for glycaemic control. If a patient is on a stable oral regimen with HbA1c concentration

blood sugar, such as driving and handling dangerous machinery

anxiety about one's blood sugar control and state of health

insulin-treated patients who were able to adjust their regimen. [3]

the physical pain of finger pricking

expense to the NHS or other medical body

It is important to point out however that this debate is not straightforward, however. One factor that is often overlooked is that the numbers obtained by testing are only one part of the picture, which requires additional data to be complete. For example, it is important to relate the number to what and when the patient last ate. The patient's exercise regime must also be considered, as well as when and how much medication has been taken. If this were not complicated enough, the patient (and physician) need to consider factors such as whether the patient has recently been ill, or even subjected to high levels of stress, which can distort the picture.

Blood glucose meters which utilise an enzyme electrode (a term first coined by Updike and Hicks in the late sixties [6]) as the glucose sensing element are particularly suitable medical devices for SMBG.

All the commercial electrochemical meter systems typically comprise two components. The meter - applies potential differences in a programmed sequence to the sensor, collects current data and analyses the current time response of the sensor, records and displays results. The enzyme electrode (or biosensor) test strip - collects the blood sample, the sample undergoes an enzymatic chemical reaction followed by an electrochemical detection step. The patient simply inserts the enzyme electrode into the meter and applies a small drop of blood to the sensor. After a short delay (typically 5 seconds) the blood glucose value is displayed by the meter in mg/dL or mmol/L.

The advantages offered by biosensors in SMBG arise for the following reasons. Blood is a complex fluid and glucose levels vary widely over time in a single patient, many factors besides glucose vary in blood from healthy, patients (haematocrit, oxygen levels, and metabolic by-products) therefore great specificity is a prime requirement. In addition, patients with diabetes may have a wide range of other medical problems creating even greater variation in their blood. Finally, biosensors can be used directly in the blood without requiring major modifications to the biological sample (increased temperature or pressure, dramatic pH changes, addition of highly reactive chemicals, etc).

The enzyme electrodes commonly used in SMBG can be defined as a combination of any electrochemical probe with a thin layer of enzyme based reagent that is selective for glucose (*or other important analytes such as β-hydroxybutyrate*). In these devices, the function of the enzyme is to provide selectivity by virtue of its biological affinity for a particular substrate molecule. For example, an enzyme is capable of catalysing a particular reaction of a given substrate even though other isomers of that substrate or similar substrates may be present.

Typically, the progress of the enzyme reaction (which is related to the concentration of analyte) is monitored by the rate of formation of product or the disappearance of a reactant. If either the product or reactant is electroactive, then the progress of the reaction can be monitored directly using amperometry. In this technique, current flow is measured in response to an applied voltage. The resultant current is monitored by the meter and then interpolated into an accurate measurement of glucose using on-board software algorithms giving the user a concentration value in typically less than 7 seconds.

Amperometric Glucose Sensors for Whole Blood Measurement Based on Dehydrogenase Enzymes 323

functionalities on the surface of the electrode and second the reaction product, NAD+, adsorbs onto the electrode surface. Both of these processes are irreversible and result in the gradual blocking of the electrode during continued oxidation. It is this electrode fouling that results in the irreproducibility of the analytical signal from bare electrodes. In addition, if this method were to be used in commercial glucose sensors for SMBG, the high over potentials required would result in the response being a combination of signals from the oxidation of both NADH

The problems mentioned above can be overcome by using small catalytic molecules called mediators. These molecules can transfer charge from the reduced coenzyme directly to the electrode thereby bypassing the direct oxidation of NADH. The use of this approach has three potential advantages when compared to the direct oxidation of the reduced nicotinamide. First, by judicious choice of the mediator problems associated with electrode fouling or competing reactions can be avoided (especially true if he chosen mediator has a site for hydride transfer). Virtually 100% of the oxidation product via this mechanism is biologically active NAD. Second, the rate of electron transfer between the NADH molecule and the mediator can be enhanced resulting in a more stable increased signal. The rate at which NADH is chemically oxidised will governed to a large extent by the potential difference between the formal redox potential of the two species. Finally, a mediator can be selected with a redox potential that will limit the effects of interference. This is particularly relevant in cases where the bio-analyte is present in blood at low concentrations as in the case of beta-hydroxybutyrate (0.0 – 0.3mEq/L). The electrocatalytic oxidation of NADH at a modified electrode is shown schematically in Figure 1. Of particular

and common interferents, e.g. ascorbic acid, uric acid, paracetamol etc. found in blood.

**Figure 1.** Cyclic voltammograms of the o-AP modified GC electrode in phosphate buffer (a) in the absence and in the presence of increasing concentrations of NADH/mM: (b) 0.1, (c) 0.3 and (d) 0.5; and (e) 0.6 at bare GC in phosphate buffer (0.1 M, pH 7.0). Scan rate = 10 mV/s. Note, the peak oxidation for NADH at the bare electrode occurs at 0.6 Volts. When the electrode is modified, in this case with oamino phenol the oxidation of NADH is concomitant with the oxidation of the immobilised redox

couple (traces b – d). *Figure and data adapted from reference 11.*

The final method of analysis used will ultimately depend on several properties of the enzyme. The main considerations are;

The answer to the first three criteria will depend largely on the system under investigation. The answer to the latter three depends on the requirements and application of the sensor under consideration. If the enzyme does not contain any redox groups, then the method of analysis will be restricted to monitoring either the release of products or the consumption of substrate by their reaction at the transducing electrode. The current produced can then be related to the concentration of analyte. Of particular relevance to this article, are the devices that incorporate nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD) or pyrroloquinoline quinone (PQQ) dependent dehydrogenase enzymes.