School of Biomedical Engineering and Health Sciences,

Faculty of Engineering, UTM.

SMBE 4413: Biochemistry for Biomedical Engineers

Assignment 2: Glucose Biosensor

Lecturer: Dr Norhana Binti Jusoh

Name: Lim Jia Hui

Matric Number: A16MB0077
1.0 Introduction

According to World Hearth Organization (WHO), about 422 million people worldwide
have diabetes, particularly in low-and-middle income countries. Diabetes is a chronic, metabolic
disease characterized by elevated levels of blood glucose, which leads over time to serious damage
to the heart, blood vessels, eyes, kidneys, and nerves.

The most common is type 2 diabetes, usually in adults, which occurs when the body
becomes resistant to insulin or doesn't make enough insulin. In the past three decades the
prevalence of type 2 diabetes has risen dramatically in countries of all income levels. Type 1
diabetes, once known as juvenile diabetes or insulin-dependent diabetes, is a chronic condition in
which the pancreas produces little or no insulin by itself.

Due to the need in maintaining normal blood glucose levels, a series of suitable glucose-
measuring devices have been developed. The purpose of blood glucose monitoring is to reduce the
risk of developing complications with diabetes, to allows diabetics to see if the s=insulin and other
medication they are taking are working, to give diabetics an idea as to how exercise and food affect
their blood glucose and lastly is to prevent hypoglycemia or hyperglycemia. Biosensor technology
has developed rapidly and can play a key role providing a powerful analytical tool with major
applications particularly in medicine. Today’s biosensor market is dominated by glucose
biosensors. This report review the history and the basic principles of operation of glucose
biosensors.

2.0 History of Glucose Biosensors

There are three generations of glucose biosensors from 1962 to 2000. The evolution of the
glucose biosensors will be discussed in the sections below.
 2.1 First-Generation Glucose Biosensor

The concept of glucose biosensor was proposed by Clark and Lyons in 1962. This glucose
biosensor was composed of an oxygen electrode, an inner oxygen semipermeable membrane, a
thin layer of GOx, and an outer dialysis membrane. Enzymes could be immobilized at an
electrochemical detector to form an enzyme electrode. A decrease in the measured oxygen
concentration was proportional to the glucose concentration. Updike and Hicks significantly
simplified the electrochemical glucose assay by immobilizing and thereby stabilizing GOx. They
immobilized GOx in a polyacrylamide gel on an oxygen electrode for the first time and measured
glucose concentration in biological fluids. It estimated glucose concentrations in the sample based
on hydrogen peroxide production by glucose oxidase utilizing dissolved oxygen. The operation of
the glucose biosensor is shown in Figure 2.1 below.

Figure 1 Principle of First-Generation Glucose Biosensor

Based on this technology, Yellow spring Instrument company launched the first
commercial glucose biosensor in market in 1975 for the direct measurement of glucose. The first
generation glucose biosensor were based on the use of natural oxygen substrate and on the
detection of the hydrogen peroxide produce. Measurements of peroxide formation have the
advantage of being simpler, especially when miniature devices are being considered. However, the
main problem is the amperometric measurement of hydrogen peroxide required a high operating
potential (0.6V) for high selectivity. The restricted solubility of oxygen in biological fluids will
produce fluctuations in the oxygen tension and also the hydrogen peroxide produced will
deactivate the enzyme.
 2.2 Second-Generation Glucose Biosensor

The second-generation glucose biosensor is the improved version of first-generation

glucose biosensor. It utilized redox mediator to transfer electrons form the enzyme to the working
electrode surface. The principle of second-generation glucose biosensor is shown in Figure 2.2
below.

Figure 2.2 Principle of Second-Generation Glucose Biosensor

A variety of redox mediators such as ferrocene, ferricyanide, quinines, methylene blue

were used to improve the performance of the sensor. The usage of the redox mediator will
eliminate the need of oxygen for electron transfer at the electrode surface, thus overcoming the
drawback of limited oxygen pressure observed in the first generation glucose biosensor. The lower
redox potential of chosen mediators which is 0-2V results in no reference from other electroactive
species such as uric acid or ascorbic acid. In addition, the redox mediator will enhance the electron
transfer between the redox center of enzyme and the electrode surface.

Despite the advantages of the second-generation glucose biosensor, it had some drawbacks.
There was high competition between redox mediator and oxygen and the interference of other
electroactive species lead to false and inaccurate results.
 2.3 Third-Generation Glucose Biosensor

The third-generation glucose biosensors are reagentless and based on direct transfer
between the enzyme and the electrode without mediators. Instead of mediators with high toxicity,
the electrode can perform direct electron transfers using organic conducting materials based on
charge-transfer complexes.

The intrinsic barrier to electron flow is the globular structure of glucose oxidase with the
active site containing FAD/FADH2 redox cofactor, buried deep inside a cavity of ~ 13 Aº is a major
hinderance for direct electron transfer. The carbon nanotubes immobilized the electrode surface
provide the suitable orientation for enzyme immobilization and establish connection between
electrode surface deeply. The principle of third generation glucose biosensor is shown in Figure
2.3 below.

Figure 2.3 Principle of Third-Generation Glucose Biosensor

3.0 Basic Principle of Glucose Biosensors

A biosensor composes of three main parts, the biological recognition elements that
differentiate the target molecules in the presence of various chemicals, a transducer that converts
the biorecognition event into a measurable signal, and a signal processing system that converts the
signal into a readable form. The molecular recognition elements include receptors, enzymes,
antibodies, nucleic acids, microorganisms and lectins. The five principal transducer classes are
electrochemical, optical, thermometric, piezoelectric, and magnetic. The majority of the current
glucose biosensors are of the electrochemical type, because of their better sensitivity,
reproducibility, and easy maintenance as well as their low cost. Electrochemical sensors may be
subdivided into potentiometric, amperometric, or conductometric types. Enzymatic amperometric
glucose biosensors are the most common devices commercially available and have been widely
studied over the last few decades. Amperometric sensors monitor currents generated when
electrons are exchanged either directly or indirectly between a biological system and an electrode.

The basic concept of the glucose biosensor is based on the fact that the immobilized GOx
catalyzes the oxidation of β-D-glucose by molecular oxygen producing gluconic acid and
hydrogen peroxide [35]. In order to work as a catalyst, GOx requires a redox cofactor—flavin
adenine dinucleotide (FAD). FAD works as the initial electron acceptor and is reduced to FADH2.

Glucose + GOx − FAD+ → Glucolactone + GOx − FADH2

The cofactor is regenerated by reacting with oxygen, leading to the formation of hydrogen
peroxides.

GOx − FADH2 + O2 → GOx − FAD + H2 O2

Hydrogen peroxide is oxidized at a catalytic, classically platinum (Pt) anode. The electrode
easily recognizes the number of electron transfers, and this electron flow is proportional to the
number of glucose molecules present in blood [36].

H2O2 → 2H+ + O2 + 2e
 Three general strategies are used for the electrochemical sensing of glucose; by measuring
oxygen consumption, by measuring the amount of hydrogen peroxide produced by the enzyme
reaction or by using a diffusible or immobilized mediator to transfer the electrons from the GOx
to the electrode. The number and types of GDH-based amperometric biosensors have been
increasing recently. The GDH family includes GDH-pyrroquinolinequinone (PQQ) and GDH-
nicotinamide-adenine dinucleotide (NAD) [40–42]. The enzymatic reaction of GDH is
independent of the dissolved oxygen. The quinoprotein GDH recognition element uses PQQ as a
cofactor.

Glucose + PQQ(ox) → gluconolactone + PQQ(red)

This mechanism requires neither oxygen nor NAD+. GDH-PQQ is a particularly efficient
enzyme system, with a rapid electron transfer rate, but it is relatively expensive.

GDH with NAD as a cofactor produces NADH rather than H2O2. NAD is a major electron
acceptor in the oxidation of glucose, during which the nicotinamide ring of NAD+ accepts a
hydrogen ion and two electrons, equivalent to a hydride ion. The reduced form of this carrier
generated in this reaction is called NADH, which can be electrochemically oxidized.

Glucose + NAD+ → gluconolactone + NADH

NADH → NAD+ + H+ + 2e