U.A. NADKARNI MSc THESIS - Copy

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U. A. Nadkarni

Health Tracker: Biosensors

SCHOOL OF HEALTH

Medical Diagnostics

MSc THESIS

Academic Year 2013 - 2014

Supervisor: J. D. Newman

September 2014

This thesis is submitted in partial fulfilment (40% weighting) of the

Requirements for the degree of Master of Science

© Cranfield University 2014. All rights reserved. No part of this publication

May be reproduced without the written permission of the copyright owner.

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Abstract

In this project Health Tracker : Biosensors attempt is made to rationalise the meaning of

wellbeing and various health tracker parameters by which this health wellbeing can be

assessed is discussed. The importance of changing trend of diseases and morbidity and

mortality worldwide both in developed & developing world is projected and further attempt

is made to reason out why chronic condition such as Diabetes Mellitus needs to be tackled

head on. The importance of Biosensor in health tracking the blood glucose check regularly

and proposed methods and laboratory method used is rationalised.

Finally the actual laboratory experiments carried out, the results produced and their

interpretation is discussed and conclusions drawn to complete the project.

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Acknowledgements

I am grateful for all the help I received from my supervisors, Dr Jeffrey Newman and

Professor Selwayan Saini, Inside Biometrics Ltd for their support and guidance throughout

my dissertation.

I would also like to give a special thanks to my family, especially my mother and my father

for their continued support and encouragement throughout my studies.

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Table of Contents

Page

Abstract 2

Abbreviations 11-12

Chapter 1 Introduction and Literature Review 13

1.1 General Introduction 13-14

1.2 Assessment of Wellbeing (Measurement) 14-15

1.3 Methods for monitoring wellbeing 15

2.0 Health trackers (Prioritized List) 15

2.1 Blood Glucose 15-16

2.2 HbA1c (Glycated Haemoglobin) 16

2.3 Blood RBC's & WBC's 17

2.4 Cholesterol/HDL/LDL 17-18

2.5 Blood Pressure 18-19

2.6 Electrocardiogram (ECG) 19-20

2.7 Heart Rate 20

2.8 Respiratory Rate 20-21

2.9 Blood pH 21

2.10 Body Temperature 21-22

2.11 Exercise Lactate 22

2.12 Body Mass Index (BMI) 22-23

2.13 Blood Alcohol 23

2.14 K+ (Potassium) 23-24

2.15 Drugs of Abuse 24

3.0 Opportunities for measurements of Health Tracker Parameters/Indices/Markers 25

3.1 Diabetes Mellitus 25-27

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3.2 Infectious Diseases 27-28

3.3 Malnutrition 28

3.4 Mental illness 28-29

3.5 CHD (Coronary Heart Disease) 29

3.6 Cancer 30

3.7 COAD (Chronic Obstructive Airway Disease) 30-31

4.1 Disease Prevalence and Incidence 31-40

4.2 Burden of Disease and Cost Effectiveness Estimates 40

4.3 Effectiveness of monitoring 41-42

5.1 Aims 42

5.2 Objectives 42

6.1 Monitoring Systems 42-43

6.1.1 Physiological sensors 43-45

6.2 Chemical sensors 45-46

6.3 Remote Sensors 46-47

6.4.1 Biosensors 47

6.4.2 Market & Applications 48

6.4.3 The Basic Components of Biosensors 48-49

6.4.4 Requirements of Biosensors 50

6.4.5 Transducer/ Bio transducer 51

6.4.6 Functioning of Biosensor 51

6.4.7 Classification of Biosensors 51-52

6.5 Glucose Biosensors 52-53

6.5.1 A brief historical perspective of glucose sensing 53

6.5.2 Generations of Biosensors 53

6.5.3 First Generation Glucose Biosensors 54

6.5.4 2nd Generation Glucose Biosensors 54

6.6.1 Problems to surmount in Glucose Biosensors 54

6.6.2 Enzyme Interferences 55

6.6.3 Electrode Interference 55

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6.6.4 Fouling 56

6.7.1 Measurement by finger pricking (Point of Care) 56

6.7.2 Continuous Measurement of Glucose 56-57

6.7.3 Non-invasive Glucose Measurements by sensors 57

6.8.1 Enzymes and their mechanisms of action 57-58

6.8.2 Methods of Immobilisation of Enzymes (with Advantages and disadvantage58

6.8.3 Physical Adsorption 58

6.8.4 Enzyme Entrapment 58

6.8.5 Micro Encapsulation 59

6.8.6 Cross Linking 59

6.8.7 Covalent Bonding 59

6.9 Various uses of Enzyme (Glucose Oxidase GOx) 59-60

Chapter 2 Materials & Methods 61

1.1 Materials & Methods (for Electrochemical Biosensors) 61

1.2 Biological Recognition Element 61

2.1 Material for Electrodes and Supporting Substrates 61-62

2.2 Membrane Materials 62

2.3 Materials for immobilisation of Biological Recognition Elements 62-63

3.1 Methods for Fabrication 63

3.2 Electrode Fabrication Methods in current use 63-64

3.2.1 Screen Printing 64

3.2.2 Deposition Method 64

3.2.3 Polymerisation 64

3.2.4 Plasma Induced Polymerisation 64

3.2.5 Photolithography 65

3.2.6 Nanotechnology 65

Chapter 3 Rationalisation 66

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1.1 My proposed Method for the Glucose measuring Device and Schematic plan (What

can we do better?) 66

1.2 Fabrication of Electrode (working electrode) 66

1.3 Construction of outer membrane 67

2.1 Rationalisation 67-70

2.2 History of Glucometer 71

Chapter 4 Design of Device 72

1.1 Apparatus – Electrodes Construction 72

1.2 Reference Electrode 72

1.3 Counter Electrode 72

1.4 Enzyme Electrode Construction 73

1.5 Construction of Membrane layer 73

1.6 Preparation of Buffers solution 74

1.7 Preparation of Glucose Solutions for Testing 74

1.8 Experimental Method and Application of Membranes 74-75

1.9 Equipment used 75-76

Chapter 5 Results from Device 77

1.1: Sensor 1 Buffer 77

1.2: Sensor 1 1mM 77

1.3: Sensor 1 5mM 77-78

1.4: Sensor 1 10mM 78

1.5: Sensor 1 100mM 78-79

1.6: Concentration Vs Current : Sensor 1 79

1.7: Sensor 2 Buffer 80

1.8: Sensor 2 1mM 80

1.9: Sensor 2 5mM 80-81

1.10: Sensor 2 10mM 81

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1.11: Sensor 2 100mM 81-82

1.12: Concentration Vs Current: Sensor 2 82

Chapter 6 Discussion / Conclusions 83

References 84-87

Appendices 88-91

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List of Tables

Table 1 : Prevalence (millions) of selected conditions by WHO region; 2004 32

Table 2 : Incidence in (millions) of selected conditions by WHO region; 2004 34

Table 3: Incidence in (millions) of selected conditions by WHO region; 2004 39

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List of Figures

Figure 1: The major waves of a single normal ECG pattern 20

Figure 2 : Worldwide distribution of diabetes prevalence in 2005 and estimated numbers for

2030 (adapted from WHO, 2004) 25

Figure 3: The 10 leading causes of death in the world; WHO (2014) 35

Figure 4: The 10 leading causes of death in the world by percentage; WHO (2014) 36

Figure 5: The 10 leading causes of death in the United States in 1900 and 1997; 2014 38

Figure 6: Mote1 Form factor; Sensor: ECG, Temperature, GSR, Accelerometer 43

Figure 7: Mote 2: Form factor; Sensor: Respiratory Inductive Plethysmograph (RIP) 44

Figure 8: Bridge Mote Form Factor 44

Figure 9: End – to – End System 44-45

Figure 10: Signal Captured from the Autosense System on a laptop appears below: Red is

Respiration and Green is the ECG. 45

Figure 11: Composition of Chemical Sensor 46

Figure 12: A LIDAR (Light Detection and Ranging ) Image created with data collected by

NOAA’s National Geodetic Service 47

Figure 13 Schematic diagram showing the main components of a biosensor 49

Figure 14: Working principles of a Biosensor 49

Figure 15: Glucose Detector 49

Figure 16: Principles of different enzyme immobilisation methods (adapted by Turner.et.al;

1987) 58

Figure 17: Anton H. Clemens (right) was the inventor of the first blood glucose meter while

he was the director of the Ames Instrument R&D Department of the Ames Division of Miles

Laboratories Inc. in Elkhart, Indiana. Here he is shown talking to George W. Orr, Jr., Group

Vice President, Professional Products Group (left) and Walter Ames Compton, M.D.,

President and Chief Executive Officer of Miles Laboratories Inc. (centre). This photograph

appeared in the 1969 Annual Report of Miles Laboratories Inc.; Mendoza, D. (11/1999),

Blood Glucose Meters, available at: www.mendosa.com/bgmeters.htm (accessed 07/2014).

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Abbreviations

CDC Centres for Disease Control & Prevention

WHO World Health Organization

PSI Public Services International

WHO World Health Organization

HIA Health impact Assessment

DALY Disability Adjusted Life Year

EIA Environmental Impact Assessment

HIA Health Impact Assessment

AIDS Acquired Immunodeficiency Syndrome

HIV human immunodeficiency virus

STD Sexually Transmitted Diseases

NHIS National Health Interview Survey

HRQOL Health-Related Quality of Life

C6H12O6 Glucose

C2H5OH. Alcohol

ECG Electrocardiogram

BMI Body Mass Index

HbA1c Glycated Haemoglobin

LDL Low density Lipoprotein

HDL High density Lipoprotein

VLDL Very low Density Lipoprotein

NHLI National Heart & Lung Institute

GABA Gamma amino butyric acid

PET scan Positron Emission Tomography

NIDA National Institute on Drug Abuse

BAC Blood Alcohol Concentration

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CHD Coronary Heart Disease

COAD Chronic Obstructive Airway Disease

GBD Global Burden of Disease

MPKB The Marshall Protocol Knowledge Base

SCR Skin conductance Response

GOx Glucose Oxidase

H2O2 Hydrogen peroxide

KCl Potassium chloride

FAD Flavine adenine dinucleotide

NAD Nicotine adenine dinucleotide

GDH Glucose dehydrogenase

PQQ Pyrroloquinoline quinone

ISE Ion-selective electrode

ISF Interstitial fluid

Pt Platinum

SD Store Data

TBATS TetrabutylammoniumToluene Sulphonate

POC Point of Care

FET Field Effective Transistors

AFM Atomic Force Microscopy

PEG Polyethylene Glycol

ARM Ames Reflectance Meter

HEC Hydroxylethyl cellulose

BGM Blood Glucose Monitoring

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CHAPTER I: INTRODUCTION AND LITERATURE REVIEW

1.1 General Introduction:

According to World Health Organization (WHO) Health is defined as "A State of complete

physical, mental and social well-being and not merely the absence of disease or infirmity" the

correct bibliographic citation for this definition is Preamble to the constitution of the World

Health Organization as adapted by the International Health Conference, New York 19-22

June, 1946; signed on 22nd

July 1946 by the representatives of 61states and entered into force

on 7th

April 1948( As shown in official records of the World Health Organization no2 p. 100)

reproduced : http://www.who.int/about/definition/en/print.html

There has been no change or modifications to date to this definition, though there are moves

recently in that direction. WHO has stated that "The enjoyment of the highest attainable

standard of Health is one of the fundamental rights of every human being without distinction

of race, religion, political belief, economic or social conditions" reproduced: PSI ,

http://www.world-psi.org . In 21st Century apart from absence of disease or illness, health

has to take into consideration other factors such as to person's emotional, social and mental

factors ultimately connected to his or her happiness and life satisfaction, thus broadening the

view. Even according to Centres for Disease Control & Prevention (CDC), healthy mind and

healthy body together help in Health promotion and naturally increase a sense of individuals

other economically related benefits. This also increases his/her longevity of life, and person's

productive contribution and connectivity to society.

All this contributes to National wellbeing. Attempts have been made by researcher in

Australia & America to measure National Health but it is difficult though it is important to

realise that higher levels of well-being are more productive with positive contribution to

societies as seen in Developed world.

Public Services International (PSI), a global trade union represents 20 million workers

delivering vital services in 150 countries. In an article published on PSI "Health is a public

good and ill health is a fundamental humanitarian problem with political, economic and

social causes and consequences" reproduced: PSI, http://www.world-psi.org. In general

higher levels of wellbeing is seen more in economically developed countries. They have

more effective and accountable governments with lesser degree of corruption and manage

their citizen's needs of food & health.

http://www.who.int/about/definition/en/print.html

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The world Health Organisation (WHO) recognised the need for developmental policies. To

promote rather than compromise health to reduce the negative health impact it advocated

inclusion of Health component in its environmental impact assessment. There is also

included Health impact Assessment (HIA) (CooperWeil.et.al 1990). Unfortunately Health

Professional; who should really be an integral part of any development and may thus find it

uncomfortable to participate in EIA process e.g. building highway through very thinly

populated area may not require much health planning input but if a large dam is to be

constructed passing through densely populated subtropical countries with high levels of

AIDS and vector borne diseases, like malaria, it would need expertise in environmental

health & health promotion. Hence health impact assessments include integrated steps of

review, research, socio demographic assessment to assess population at risk, health

determinant assessments (water & sanitation, food, housing, fuel security, pollution, public

infrastructure, waste management etc. which affect health.

The other factors included are Health status assessment such as quantitative assessment in

terms of morbidity & mortality due to diseases, conditions & injuries; and World Bank has

brought it out (WHO 1996) in terms of Disability Adjusted Life Year (DALY) discussed

under section 4.3. The other assessments need to be done are regarding Health Services

deliveries in terms maternal & child health promotion, family planning, STD clinics,

Hospitals, community Health workers etc. This really is a gigantic task but HIA planning

needs to be really good if populations are to secure their wellbeing in terms of housing, job

satisfaction, social connectivity, neighbourhood security etc. mentioned above.

Unfortunately developing countries rather than developed ones, who need this planning, with

their limited resources and local EIA and HIA planning facilities have more problems in

coping with this task.

1.2 Assessment of wellbeing (measurement)

Wellbeing is difficult to quantify and thus difficult to measure as it is more subjective

assessment by an individual himself of his own condition and expressed in his own words.

However NHIS (National Health Interview Survey in 2001- NHIS 2001) was carried out

using also some objective parameters such as individual satisfaction regards his basic needs

of job & income, housing, neighbourhood, social relationship etc. The combination of these

subjective and objective indices which are called Psychometric & utility based techniques is a

more useful method of measurement. To assess perhaps the National wellbeing the index

was placed between 0(death) to 1(optimum health) at the two extreme points. On this scale

males or females between ages of 20-39 years measured significantly better wellbeing (scores

≥0.82) compared to males and females 40 years or older (scores ≥0.79) . Reproduced: CDC,

(Health-Related Quality of Life (HRQOL) Wellbeing Concepts

http://www.cdc.gov/hrqol/wellbeing.htm).

Data from 2005 Behavioural Risk Factor Surveillance Electrochemical biosensors:

Recommended definitions and classification Thévenot, D.R., Toth, K., Durst, R.A., Wilson,

G.S. 2001 Biosensors and Bioelectronics 16 (1-2), pp. 121-131system(BRFSS) in USA

http://www.cdc.gov/hrqol/index.htm

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demonstrates 5.6% of US adults were dissatisfied/very dissatisfied with their lives but Porter

Novelli Health style data (2008) reveals 30% adults most days feel sense of accomplishment

from what they do. Reproduced: CDC, (Health-Related Quality of Life (HRQOL) Wellbeing

Concepts http://www.cdc.gov/hrqol/wellbeing.htm).

Apart from National level, at individual level genetic factors, personality and demographic

come into play. In general there is found to be U-shaped distribution of wellbeing age wise.

Younger and older adults to have more wellbeing compared to middle-aged individuals who

are subject to more stress and have responsibilities still to fulfil. There is now thus a move

towards measuring the health of individuals in more holistic way (physical + mental health)

Reproduced: CDC, Health-Related Quality of Life (HRQOL) Wellbeing Concepts

http://www.cdc.gov/hrqol/wellbeing.htm.

Incidentally mental health is defined as "a state of wellbeing in which every individual

realises his or her own potential, can cope with the normal stresses of life, can work

productively and fruitfully and is able to make a contribution to her or his community".

Reproduced: CDC, Health-Related Quality of Life (HRQOL) Wellbeing Concepts

http://www.cdc.gov/hrqol/wellbeing.htm. There is therefore now a move at WHO to broaden

the definition of Health. There has also been an attempt to measure the health at National and

International levels. Such attempts have been made in Australia and in America but it is

difficult due to obvious gigantic nature of the task though the attempts are further continuing

in the matter.

1.3 Methods for Monitoring Well-Being

With advances in physics and chemistry and various subspecialties such as electrochemistry,

optical physics e.g. Raman Spectroscopy, especially in the field of nanotechnology the things

have taken a quantum jump in the quantitative assessment of health monitoring systems in

medicine & other fields. In medicine in particular ability has now been achieved to measure

various parameters/indices pertaining to individual health and I shall discuss some of them

briefly in the discussion that follows.

I shall now underneath mention some of the body's indices/parameters/markers that are

routinely measured by Health Tracker and discuss them briefly.

2. Health Trackers (Prioritized List):

2.1 Blood Glucose:

All the cells in our bodies need for physiological processes or metabolism energy which is

supplied by Glucose transported in blood. The glucose is produced from all the food we eat.

The human body normally regulates blood sugar levels strictly within a narrow range of 4-

7mmol/80-120mg so as not to be too high or too low in order to maintain stability and



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equilibrium of internal environment called Homeostasis. Post prandially (after 2 hours of

meal), the normal variation is ≤140mg/dl (7.8mmol/l). With passing age e.g. after 60 this

level is a little higher 8.9mmol or ≤160mg/dl due to increasing glucose intolerance.

Sugar or glucose is a simple form of carbohydrate body make into, prior to use by cells

particularly brain cells which need steady supply of glucose (C6H12O6). The Glucose

however can enter cells only in the presence of Hormone (Insulin) secreted by the βcells of

pancreas, an abdominal organ. The release of this hormone in the body is controlled by what

is called feedback mechanism with supervisional control by central nervous system. In the

healthy body excess glucose is converted to Glycogen (a form of polysaccharide) by liver and

muscles. Derangement of this normal control mechanism by either autoimmune destruction

of βcells of Pancreas or a condition of Insulin resistance results in a pathology of chronic

hyperglycaemia (called Diabetes Mellitus). If the blood sugar is excessive while passing

through kidney it may not be absorbed being thrown out in urine producing what is called

Glycosuria. On the other hand tumour of βcells called Insulinoma or more commonly excess

or erroneous intake of antidiabetic medication may result in low blood sugar called

hypoglycaemia which is much more serious. A stage of Biochemical or absolute

Hypoglycaemia is reached when blood sugar falls to below 2.2 milli moles/litre. This can

cause serious damage to all but more important to central nervous system and life

threatening.

Various forms of blood glucose estimations are done including Fasting blood glucose,

Random sample, postprandial (2 hours after meal), and Oral glucose tolerance especially in

pre- diabetics depending on indication. For overall review of maintenance of Diabetic

control Glycated Haemoglobin (called HbA1c) measure which is popular test at present

described below. Ref: Diabetes Health Centre: Blood Glucose;

http://www.webmd.com/diabetes/blood-glucose

2.2 HbA1c (Glycated Haemoglobin):

As stated above this test is routinely used in Diabetics to evaluate Glycaemic control in

previous 6-12 weeks and thus to adjust medications and other measures in conjunction with

home blood glucose monitoring. The normal range of glycated Hb is between 4% to 5.6%.

Between 5.6% - 6.4% there is increased risk of Diabetes. In known diabetics aim is usually

to maintain level to at least less than 7%. The normal lifespan of RBC's is approximately 120

days and they especially in their latter span glycate excess glucose. Hence this gives idea of

Diabetic control in previous 6 - 12 weeks. Higher levels of HbA1c leaves the risk of

Diabetes related complications of cardiovascular conditions of Heart disease and strokes.

In Diabetics regular 3 monthly checks are required but in well controlled ones, 6monthly

check-ups of HbA1c suffice.

Anaemia, liver and kidney disease, high cholesterol levels and Vitamin C adversely affect

results of this test. Ref: Diabetes Health Centre: The Haemoglobin A1c (HbA1c) Test for

Diabetes http://www.webmd.com/diabetes/guide/glycated-hemoglobin-test-hba1c

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2.3 Blood RBC's & WBC's

RBC's, WBC's & Platelets are cellular components of blood the fluid component being

plasma; RBC's & WBC's & Platelets are formed in the marrow of the bones namely the red

marrow usually from the flat bones such as ribs, skull bones, breast bone(Sternum), hipbones,

and thigh bones in early childhood when red marrow still exists. The normal RBC count is

approximately 5 million/cc in males & 4.5 million/cc in females. The corresponding figure

of normality for WBC being (4.0 -11.0/cc). Low Oxygen Tension or Hypoxia e.g. at high

mountains or anaemia (Low RBC count) is a stimulus for production of more RBC's, the

other factor being Erythropoietin a hormone produced by the kidneys. The main function of

RBC's is to carry O2 from lungs to tissues by loosely binding it to specialised protein in it

called Haemoglobin and after dissociation of this O2 in tissues collect CO2 from them by use

of enzyme called carbonic anhydrase and to deliver it to lungs through capillaries for loss by

exhalation. The peculiar biconcave shape of RBC's and it's a nucleate nature in matured

RBC's in blood makes it more efficient in this process.

The WBC's on the other hand perform the function of Defence and immunity and are

nucleate in nature. Normally of different types including granulocytes e.g. neutrophils,

eosinophils, basophils and others such as lymphocytes and monocytes involved in immunity

& phagocytosis of larger particles. Neutrophils are increased in acute infections, eosinophil's

in allergic & worm infestations. The normal life span of RBC's is 120 days; that of WBC's

varies from 24 hours to many days depending upon the nature of the cell. The normal RBC's

and WBC's counts are a sign of good health. Ref: how stuff works "Red Blood Cells"

http://health.howstuffworks.com/human-body/systems/circulatory/blood1.htm

Having enumerated some of the health tracker (parameters/indices/markers), I believe it

would be entirely be appropriate to mention in brief common conditions met with in routine

practice (individually, nationally and internationally) where measuring these

parameters/indices/markers prove vital and help immensely in finding relief and control for

conditions. I shall mention these opportunities below. It is also important to study the

prevalence and incidence of disease, changes in the pattern of conditions which used to be top

killers in the past 2 centuries and current trend in conjunction with burden of disease and cost

effectiveness to realise the impact on wellbeing and the importance of measuring

effectiveness of monitoring. This is particularly so because in this jet age millions of people

travel to and fro and communicate globally. The advent of internet and TV also makes

people more aware and anxious about the matters and the concept of global village makes it

fully appropriate.

2.4 Cholesterol/HDL/LDL:

This is a wax like fatty substance required in the body in the production and maintenance of

cell walls or membranes, Vitamin D and bile acids required in the digestion of fat. It also is

required for the production of hormones such as steroid hormones. It is produced in the body

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by liver and other cells. The external important food sources are eggs, meat and dairy

products including milk and its derivatives. Though important, body needs cholesterol in

only small quantities. Excess of it in blood however causes deposition on the inner lining of

vessel walls in the form of hard waxy layer called Plaques. Over the years it leads to

narrowing and hardening of blood vessel with impediment to blood flow especially in

Diabetics in whom the cholesterol levels tend to remain high. The serious consequences such

as chest pain (Angina), Heart attack (myocardial infarction) or cerebral strokes can follow

depending on the blood vessels involved and level of involvement. These situations cost

considerably not only to affected individual but also to the family, society and government as

such in terms of monitory, social, productivity, morbidity and mortality. Hence the

importance of maintaining normal levels of Blood cholesterol. Cholesterol travels in blood

attached to protein; this combination is called Lipoprotein and depending on the amount of

proteins in relation to fat there are normally 3 types of Lipoproteins:

i. Low density Lipoprotein (LDL) : - called bad cholesterol resulting in plaques

ii. High density Lipoprotein (HDL): - good cholesterol as lower risk of Heart disease

iii. Very low Density Lipoprotein (VLDL): - mostly fats & non proteins

Excess of fats, calories; alcohol etc. get converted to (iv) triglycerides or simply stored as fat.

The normal LDL values remain between 100 - 129mg/dl. Above 160/dl are considered high.

Similarly for Triglycerides<150mg/dl are considered normal, above 200 mg/dl high

Factors such as diet (high fat diet), Diabetes, age, gender and overweight and, smoking affect

cholesterol levels adversely. On the other hand, good exercise, low fat diet and medicines

such as statins lower the cholesterol levels. Ref: Cholesterol & Triglycerides Health Centre;

The BasicsofCholesterol http://www.webmd.com/cholesterol-management/guide/cholesterol-

basics; Cholesterol & Triglycerides Health Centre: http://www.webmd.com/cholesterol-

management/guide/understanding-numbers

2.5 Blood Pressure:

Blood pressure is the force of blood pushing against the artery walls each time the heart

pumps the blood into the vessels to supply organs and tissues. Its importance lies in the fact

that it is the pressure against which heart has to pump and exert. Hence High blood pressure

strains the heart. Usually this is associated with narrowed blood vessels by cholesterol

plaques as mentioned above under cholesterol and can lead to heart attacks and strokes.

The blood pressure can be measured by doctors or nurses traditionally by using mercury

manometers cuff and stethoscope. This gives upper and lower readings called systolic and

diastolic blood pressures. Systolic is normally a reading produced by contraction of the heart

(systole) and diastolic by its relaxation (diastole - filling phase). Normal values of blood

pressure as advised by National Heart & Lung Institute (NHLI) are 120/80mmHg, 120-129

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systolic - Prehypertension, 80 - 89 diastolic prehypertension (140systolic & above and 90 &

above diastolic Hypertension). It is the diastolic blood pressure which gives idea roughly

about the duration of Hypertension and also the condition of vessels and associated macro

vascular risks.

Hypertension usually is of two types due to unknown cause (essential or idiopathic &

secondary follow causes e.g. Kidney disease, disease of the adrenal glands etc.

The blood pressure can be affected by various factors such as Age, gender, emotional status,

time of the day, exercise, rest, smoking and drinking habits, high salt diet, Diabetes, High

Cholesterol in Afro Americans, etc... Hence its regular check by a common man at home

using aneroid and digital barometer to avoid risk of mercurial manometers, is important in

maintaining it, as well as taking repeat readings on different occasions before stamping

somebody as hypertensive is important.

Ref: High blood pressure (hypertension)

http://www.nhs.uk/conditions/blood-pressure-(high)/Pages/Introduction.aspx

2.6 Electrocardiogram (ECG):

This is a test, as the name suggests, checks the problems with electrical activity of the heart.

The ECG translates this electrical activity into a line tracing on a special paper. The spikes

and dips in the line tracings are called waves and are denoted by the alphabets PQRST. The

heart is a muscular pump made up of 4 chambers. Upper two are called atria and lower two

ventricles. A natural electrical system in the heart makes it contract and pump the blood into

lungs and rest of the body. An electrocardiogram is done using usually a portable machine

with a set of wires called leads that are connected to various parts of the limbs and chest to

get tracing from different angles of heart. Usually a 12 lead tracing is obtained. In hospital

sometimes a continuous monitoring is done by ECG what is called Telemetry. Tracing of

ECG gives idea about its rate (normal 60 - 100 beats per minute) or too fast or too slow as

well as whether it is regular or irregular.

As stated above ECG can be done to check heart's electrical activity to know unexplained

cause of chest pain (e.g. myocardial infarction, angina or inflammation of sacs of heart

(pericarditis). It is also done to note cause and type of irregular heartbeats (Palpitations),

shortness of breath or dizziness, to know whether heart walls are thickened (Hypertrophied),

the effectiveness of heart medicines and their side effects (toxicity) can also be assessed by

ECG, as well as the working of implanted mechanical devices (e.g. Pacemakers) and health

of the heart in disease conditions such as cigarette smoking, diabetes or high blood pressure.

Ref: Heart Disease Health Centre: Electrocardiogram http://www.webmd.com/heart-

disease/electrocardiogram

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Figure 1: The major waves of a single normal ECG pattern How to read an

Electrocardiogram (ECG). Part One: Basic principles of the ECG. The normal ECG

http://www.southsudanmedicaljournal.com/archive/may-2010/how-to-read-an-

electrocardiogram-ecg.-part-one-basic-principles-of-the-ecg.-the-normal-ecg.html

2.7 Heart Rate:

Heart rate is the rate at which the heart beats per minute. In the normal individual the heart

rate is usually given by Pulse rate felt easily by palpating at wrists (Radials), neck (Carotid),

or groins (Femoral) or over the temples (Temporal). If the heart is irregular then it may be

necessary to listen directly to chest over area of heart with stethoscope. Normal heart beats

between 60 - 100per minute at rest. The rate is easily affected by exercise, fever, emotions,

"fright or flight" and drugs such as Digoxin or β blockers as well as drug or substances of

abuse.

In the child at birth the rate is highest which slowly with growth adolescence and adulthood

settles to within normal limits. The athletes achieve slow pulse rate by their regular rigorous

training. The volume of the pulse also provides some indication about functioning of the

heart. The heart rate can also be monitored by machines.

Ref: Heart Disease Health Centre: Pulse Measurement; http//www.webmd.com/heart-

disease/pulse-measurement

2.8 Respiratory Rate:

Respiratory rate is simply the rate of breathing which is the number of breaths per minute.

The rate is assessed at rest and measured by number of times the chest rises per minute when

the person is lying down. Respiratory rate increases with fever, illness and other medical

conditions and drugs and substances of abuse. When checking respiration it is important also

to note whether the person has any difficulty in breathing. The normal respiratory rate in

adults is between 12-18 breaths per minute. Once again the normal respiratory rate in

21

humans is the highest at birth(in infancy 40 - 60/min) and growing through childhood and

adolescence settles down to adulthood rate (12 - 18/minute).

Ref: Vital Signs (Body Temperature, Pulse Rate, Respiration Rate, Blood Pressure)

http://www.hopkinsmedicine.org/healthlibrary/printv.aspx?d=85,P00866

2.9 Blood pH:

By definition PH is the inverse logarithm of hydrogen ion concentration of any given

solution. The pH of 7 is considered neutral. The normal body pH is 7.4 which is slightly

alkaline. The Hydrogen ions are extremely reactive and can affect some of the important

molecules involved in body's physiology. The normal physiology of body only functions

within a very narrow range of deviation (7.35 - 7.45). The pH change to below 6.9(severe

acidosis) and above 7.9(severe alkalosis) can have serious life threatening consequences and

are not compatible with life if the change lasts more than short while.

Thus to maintain the body's internal environment harmony, the blood has a system of buffer

pairs; namely a mixtures of two chemicals, weak acids which resist the pH change from

normality by either accepting or donating Hydrogen ion. The most important organs

concerned with this pH maintenance are lungs and kidneys. The most important buffer pair

in humans is Bicarbonate/Carbon Dioxide pair acting at the level of excretion by lungs. The

other buffer pairs are Proteins and Phosphates. The kidneys regulate Bicarbonates and some

non-volatile acids produced in metabolism through renal excretion with ultimate pH control.

Ref: Lecture Notes-39: Regulation of Blood pH Kidneys and Lungs

http://acccn.net/bio/book/bio50/lecnotes/lecnot39.html

2.10 Body Temperature:

Body Temperature is simply the temperature of the body and is an indicator of Body's ability

to generate heat under certain conditions and to get rid of it when necessary, thereby

maintaining a constant temperature for proper functioning and constancy of internal body

environment or Homeostasis. Normal temperature of body varies between (36.50C - 37.2

0C

or 97.80F - 99.0

0F) irrespective of outside temperature. Normally body maintains this

excellently through its dermal or skin surface. In hot and humid conditions there is

proportional dilatation of skin vessels. The person sweats thus, evaporation of which as well

as by radiation and convection heat is lost to the required extent.

In very cold temperatures outside, the heat is preserved by intensive constriction of skin

blood vessels and also extra heat is generated if necessary by involuntary contraction of

muscles manifest as shivering.

Above 1000F the condition is called fever which is commonly seen associated with

infections, medicines (drug fever), in women mid menstrual cycle or injury (trauma). If the

temperature rises above 390C especially in children, there can be serious consequences on

22

brain cells resulting in convulsions. At 400C and more the proteins of the cells can coagulate

resulting sometimes in an irreversible damage.

If outside temperature falls rapidly below freezing as on exposure for long time to freezing

temperatures or on immersion or drowning in freezing sea waters, the body tends to loose

heat rapidly, what is called Hypothermia.

There are 3 grades of it. Mild (350C - 32

0C); Moderate (32

0C - 28

0C) and severe (28

0C and

below) and can once again be rapidly fatal as heart tends to fibrillate and stop. The body

temperatures can be measured in a variety of ways. Orally, rectally (called core temperature),

in armpit (Axillary), from ear (Tympanic) and from skin. Ref Vital Signs (Body Temperature,

Pulse Rate, Respiration Rate, Blood Pressure)

http://www.hopkinsmedicine.org/healthlibrary/printv.aspx?d=85,P00866

2.11 Exercise Lactate:

The end product of carbohydrate metabolism in the body results in the production of energy

from Glucose with also production of energy from glucose with also product of lactic acid,

that dissociates to give rise to H+ ions, and combination of remaining compound with Na+

and K+ ions to produce salt called lactate. It was initially believed that the end product of

Glycolytic pathway was pyruvic acid which under anaerobic conditions (insufficient oxygen

during exercise) converted to lactic acid thus caused increase in lactate in blood and muscles

resulting in fatigue. The importance was naturally related to sports medicine. With progress

in research over the last 30 - 35 years the views are changing and today it is held that it is the

excess accumulation of H+ from lactic acidosis, rather than excess production of lactate itself

which is the cause of muscle fatigue and tiredness and therefore lactate is not the foe but a

friend of athletes. More research is therefore still needed. The normal range of blood lactate

is 0.5 to 2.2 millimoles/l and in exercise or sports this is thought to rise rapidly as clearance

cannot keep pace with the event and complete exhaustion occurs between 20 - 25 mmoles/l.

Ref: Lactic Acid & Blood Lactate; http://www.sport-fitness-advisor.com/lactic-acid.html

2.12 Body Mass Index (BMI)

Body mass index is a useful measure to indicate whether a person has a healthy weight

compared to her/his height. For adults the ideal BMI varies between 18.5 - 24.5 range and

above this depending on the level (25 - 29.5 - considered overweight) and (30.0 - 39.9) obese.

Above 40 person is said to be very obese. In children the BMI is interpreted differently. This

index gives an overall idea of one's health and the amount of correction required to put it

right e.g. overweight BMI makes a person more prone to conditions such as heart disease,

strokes, Diabetes, some cancers, high blood pressure and kidney disease.

Underweight BMI on the other hand (i.e. less than 18.5) manifest also with affection of health

in the form of absent periods in women (Amenorrhoea), brittle bones, iron deficiency

anaemia etc. While taking into account BMI one also needs to take into consideration other

things such as disposition (athletic, ethnicity etc.)

23

Ref: What is the body mass index (BMI) - Health Questions - NHS Choices

http://www.nhs.uk/chq/Pages/3215.aspx?CategoryID=51

2.13 Blood Alcohol:

Alcohol is chemically known as C2H5OH. It is a liquid that is commonly consumed all over

the world as a social drink. It is the only substance apart from water, which is rapidly

absorbed at the level of stomach. Hence it raises the level in the blood rapidly producing

initial stimulation of central nervous system with sense of well-being and euphoria. However

in larger doses it is depressant and impairs judgement. The effects depend upon amount of

and concentration of alcohol consumed, the rapidity of intake prior food consumption, stress

level, metabolism, apart from age, sex and weight.

As alcohol impairs judgement, drink driving is very dangerous and can lead to serious road

traffic accidents. Hence there are strict alcohol limits for drivers in civilised world and heavy

& serious penalties for exceeding the permitted limits. The legal limits by government in UK

to drivers is 35 micrograms of alcohol/ 100ml of breathe, 80mg/100ml blood and

107mg/100ml of urine. Routine police patrol checks with breathalyser test and if necessary

blood checks by Police Doctor are in place.

Apart from this there are readymade charts to assess Blood Alcohol Concentration (BAC)for

men & women which give approximate blood alcohol percentage depending on number of

drinks, for legal purposes 5 drinks are considered intoxicating and 10 & above death is

possible and there are criminal penalties. Liver detoxifies alcohol and hence in chronic

drinkers cirrhosis of liver is the end result.

Ref: BAC Charts http//www.brad21.org/bac_charts.html; the drink drive limit - GOV.UK;

https://www.gov.uk/drink-drive-limit

2.14 K+ (Potassium):

Potassium is an important, in fact vital component of intracellular compartment just as Na+

(sodium) is in the extracellular compartment. It is a mineral which is normally found in

varying amounts in all types of foods; Potassium is an electrolyte just as Na+ and Ca

++, Cl

-,

Magnesium++

are, as all these ions help to conduct electrical charges in the body. Just as

others body can only function within an in narrow range of this electrolyte and thus possesses

elaborate system of checks to maintain its homeostasis. If despite these checks, the

potassium levels go too high or too low; then it leads to complete shutdown of Nervous &

Cardiac systems, this of course is life threatening

As mentioned above regards external food resources; the green and leafy vegetables such as

spinach, crimini mushrooms, tomatoes, Vegetables especially green varieties are richest

source of potassium. Potassium and sodium exist in partnership. The dietary approach

should be to maintain good balance in Potassium & Sodium in order to maintain a good heart

and kidney health. Apart from Bananas, yoghurt and eggs are good dietary sources of

24

potassium. Normally the body maintains, along with diet a good balance of Na+ & K

+

electrolytes which helps to maintain Heart and Kidneys in good health. Conversely there is

no worry about toxicity of this ion so long as these organs are functioning well. It is in heart

failure or kidney disease that one needs to take care to prevent toxicity. It has been argued

that the diet rich in potassium helps to reduce risk of kidney stones as it prevents precipitation

of calcium in urinary system. Diarrhoea, vomiting, fluid imbalance are a common cause of

deficiency of this ion especially in Afro Asian countries with resultant morbidity and

mortality.

Ref: TheWorld'sHealthiestFoods;

http://www.whfoods.com/genpage.php?pfriendly=1&tname=nutrient&dbid=90

2.15 Drugs of Abuse:

It is important to realise the importance and seriousness of these drugs as these drugs cause

disruption to the process of normal communication between brain cells. Normally the

information collected from eyes, ears and other sensory organs is transformed into an

information that is relayed from cell to cell to certain regions of brain which specialise in

processing this information and attaching meaning and memory to it. Inside the cells the

information travels in the form of electrical signals but at intercellular gap the transmission

occurs in the form of chemical signal with the help of specialised chemicals. The process is

known as neurotransmission and the specialised chemicals are called neurotransmitters.

The process is comparable to computers & their semiconductor system except that it is much

more complex in case of brain. The drugs of abuse as mentioned above, by disrupting this

normal process alter the way a person thinks, feels or behaves. The evidence points to

induction of two important factors of drug dependence and addiction which are the main

features of organic brain disease produced by these drugs. The common known or isolated

neurotransmitters are Dopamine, serotonin, Noradrenaline, Endorphins, Gamma amino

butyric acid (GABA) etc. The drugs of abuse act in various ways of mimicking, or by

interacting with molecular components of sending and receiving process, attaching to

receptors etc. Initially as the drug leaves the system the effects wear off; intoxication ends

and the person returns to normal in initial stages of experimentation with them. In chronic

cases permanent abnormalities set in neurotransmission resulting in dependency, withdrawal

symptoms, addiction etc. The research in the field has taken impetus from availability of

brain scans currently especially from Positron Emission Tomography (PET scan).

Ref: Impacts of Drugs on Neurotransmission; National Institute on Drug Abuse (NIDA)

http://www.drugabuse.gov/news-events/nida-notes/2007/10/impacts-drugs-neurotrans...

25

3 Opportunities for measurements of Health Tracker

Parameters/Indices/Markers:

Prioritized List

As stated above I shall now briefly discuss some of the common conditions in routine

practice encountered where these measurements are appropriate in fact vital

3.1 Diabetes Mellitus:

Figure 2 : Worldwide distribution of diabetes prevalence in 2005 and estimated

numbers for 2030 (adapted from WHO, 2004)

1. This is the condition of complex endocrine metabolic disorder that results from total or

partial lack of hormone Insulin. It produces chronic hyperglycaemia or excess sugar or

blood glucose. History records show that the Egyptians first mentioned about Diabetes

1500 BC. The Greek physician called Aretaeus (130 - 20CE) named this condition

Diabetes, meaning "flowing through" as he noted the condition caused symptoms of

26

excessive urination, much thirst and weight loss. More clear documentation of Diabetes

is credited to an Arab Physician Avicenna (980 - 1037 CE) who accurately described its

clinical features, progress and above all complications. The final solution was in great

research work and isolation of insulin by Canadian physicians in 1922 by Nobel

laureates Banting and Best (Ref: History Committee Institute of Biomedical Science;

London; March 2012).

The body needs Glucose supplied by carbohydrate metabolism constantly for the

production of energy but the level needs to be maintained constantly between 4 - 6

mmoles in order to maintain internal environment of homeostasis. The excess of glucose

can usually get glycated to Haemoglobin (CHbA1c) or excess may pass down the

kidneys in urinary tract in urine called Glycosuria. Normally body can utilise glucose by

muscles and other tissues when it is allowed to enter their cells by a hormone called

insulin mentioned above. It is produced by β cells of pancreas. The autoimmune

destruction of β cells or resistance to insulin utilisation results in Diabetes Mellitus.

There are normally 2 types of Diabetes:

Juvenile or Type I Insulin Dependent as seen in some children (due to autoimmune

destruction of β cells)

Type II which is much more common (95%) manifests in adults and elderly

(predominantly due to insulin resistance)

(Ref: Type 2 Diabetes; 2nd

Edition by Andrew Krentz and Bailey)

Type III category is added by WHO to accommodate Diabetic conditions which do not

fit into above 2 types. These conditions include diseases such as endocrinopathies,

gestational Diabetes, Genetic defects etc. (Turner & Wass; 2002)

At present condition of Diabetic Disease is ubiquitous, worldwide. The condition is as

of today lifelong and incurable. As much of epidemiology and cost burdens are covered

under sections of disease prevalence and incidence as well as burden of disease and cost

effectiveness (the sections that follow) I avoid further repetition of discussion here. One

would however like to mention some important points. The most important in Diabetes

is careful monitoring and maintenance of blood glucose levels to near normal (110mg/dl

±25mg/dl) (Ref: Glucose monitoring; E. Wilkins & P. Atanosov: Med.Eng. Phys; 1996).

If glucose remains normal or at least near normal, then complications are avoidable and a

Diabetic can live an almost normal lifespan but control a must as said "Diabetes does not

go on holidays as you do"

The complications of Chronic Hyperglycaemia include macro vascular pathologies like

Coronary Heart Disease and Strokes and Microvascular entities like Nephropathy with

Renal failure. Retinopathy with blindness and Peripheral Neuropathy with attendant

ulcerations, infections etc. due to unrecognised injuries etc. Finally and most important

point, the causes of 2 types off Diabetes are different and so the treatment. Type 1

27

(Autoimmune) there is practically no indigenous insulin, complications are more due to

fluctuant blood sugars producing hyperglycaemic ketoacidosis or hypoglycaemic

episodes with attendant central nervous serious coma and convulsions. The treatment is

insulin. Either in divided doses or with open or closed loop administration. In type 2

the matter is insulin resistance (common type and in adults) and treatment is naturally

different. Diet and obesity control, exercise, oral hypoglycaemic or with insulin top-up.

Once again it brings out the importance of monitoring systems.

Above are a just a few but very important conditions in 20th

century leading into 21st

where it is almost self-explanatory the importance of health

tracker(parameter/indices/markers) with such conditions disease prevalence and

incidence as well burden of disease and cost effectiveness bring out their importance

more starkly. In this 21st century with world population explosion of nearly 6.5 billion

and increasing, scarce resources, economic instability and austerity etc. The monitoring

- measurements need to be more accurate if we are still to continue to follow the

principle of "greatest good for greatest number of people within the available resources"

3.2 Infectious Diseases:

These are the diseases basically as name would suggest are diseases that cause infections

transmitted from human to human through environment (air droplet infection) or through

contaminated water or food or excreta or through a vector e.g. Insect bite or animal.

These diseases are caused by viruses, bacteria, fungi, parasites causing spread of disease.

Infectious diseases of animals are called Zoonotic disease which can cause disease in

human if transmitted. There is huge WHO list of these diseases which cannot be written

here but to enumerate some common import diseases and to get an idea it would suffice

to mention Leprosy, malaria, Tuberculosis, Dengue fever, Hepatitis (ABCE),

Poliomyelitis, measles, chickenpox, Small pox (now eliminated by worldwide

vaccination efforts by WHO), Influenza, Cholera, Gastroenteritis, meningococcal

meningitis, HIV etc.

There are normally three distinct stages of diseases of infectious nature:

Presymptomatic

Progression or stage of clinical disease &

Outcome or stage of diminished capacity.

In the presymptomatic stage of disease is not manifest but the person is affected and

may have some vague feelings such as anorexia, malaise, mild fever etc. this is called

prodromal stage where there is an incubation period between infection and its

manifestations. Stage 2 or progression (stage of manifest disease and depending on the

disease and organs affected have different signs e.g. fever (usually high, rash, cough and

expectoration, vomiting and diarrhoea, Jaundice, or affection of brain and muscles of

extremities as in Poliomyelitis, neck stiffness in meningitis.

28

Stage 3 or Stage of Diminished capacity or morbidity & unpleasant sequelae e.g.

Pneumonia with slow recovery, permanent damage to extremities e.g. in Polio, or

dehydration with cholera. The patient may recover from immunity, timely and correct

treatment or succumb to infection with resultant mortality. In the past there have been

regular epidemics of infectious disease worldwide due to lack of knowledge, medicines

and preventive vaccinations which caused high but preventable morbidity and mortality.

But thanks to WHO efforts as well as efforts at individual national levels a great relief

has been brought. Ref: Infectious Diseases http://www.mayoclinic.org/diseases-

conditions/infectious-diseases/basics/definition/con-20033534; Epidemiology

http://gwxy.sysu.edu.cn/lxbx/english/epidemiologic%20knowledge/Selected%20Disease

%20Concepts%20in%20Epidemiology/Natural.html

3.3 Malnutrition:

Malnutrition is a condition of inadequate supply of nutrients to the body. The condition

is usually due to dietary inadequacy. Malnutrition can be of under nutrition type or over

nutrition resulting in excess supply of nutrients and resultant obesity.

This obesity is a different topic by itself and not discussed here. It is the under nutrition

which is focussed on. The causes may be many, most important reduced mobility as

seen in elderly persons, or persons suffering from disease. The person's body may not be

able to absorb the adequate amounts of nutrients due to existing disease or may be due to

economic conditions as seen in lowly developed countries.

The malnutrition may manifest in the form of loss of weight commonly losing 5-10% of

body weight in 6 months, fatigue and tiredness or proneness to infections and anaemia.

It is more widespread than one can believe. For e.g. nearly 1 in 3 persons admitted to

care homes or hospitals in UK are malnourished. There are 3 million people in UK itself

that are malnourished let alone in the developing world. The improvements can be

obtained by dietary adequacy of nutrients, and in problem cases of feeding by using

nasogastric tubes to stomach or drips of nutrients.

Malnutrition http://www.nhs.uk/conditions/malnutrition/Pages/Introduction.aspx

3.4 Mental illness

I have defined the mental health in previous discussion as per the definition provided by

WHO. To mention in short, the importance lies ( as seen in incidence and prevalence)

29

the mental illness is rapidly increasing in populations all over the world and expected to

exceed other conditions, the commonest mental disorder being depression.

The mental illness is divided for simplicity into following categories:

Anxiety Disorders - where depending on its severity patients react to even simple or

trivial matters with extreme of fright or dread

Mood Disorders - depression, mania, bipolar disorders fall in this category and especially

depression the commonest mood disorder is due to circumstances he/she faces

Psychotic Disorders - this is seen in persons suffering from hallucinations, imagining

various voices or sounds or conditions which do not exists in reality. The other

symptoms are delusions

Impulse control and addiction disorders - where the persons are driven by their craving

for things or drugs and are driven impulsively without the normal control of rationality

Eating disorders- as seen in conditions of anorexia nervosa and bulimia where persons

have revulsions for food intake.

Personality disorders - where person's behavioural attitude is that of extreme

inflexibility, rigid and irrational rather than the flexibility which society would expect of

normal behaviour

Ref: Mental Health: Types of Mental Illness http://www.webmd.com/mental-

health/mental-health-types-illness

3.5 CHD (Coronary Heart Disease)

This is a condition that results that results from affection of coronary arteries, the arteries

that supply the blood to heart muscle. As heart is beating constantly in life it needs a

constant and steady blood supply. However the lining of these vessels can get rough

ends over a period of time by deposition excess cholesterol over the vascular endothelial

lining, Diabetes, Hypercholesterolemia (High Blood Cholesterol) heredity are common

causes and smoking is a precipitating factor. The deposition results in the formation of

waxy hard material called plaques which narrow the arteries producing starvation of

heart muscle especially during exercise manifest as chest pain called angina. The patient

may be relieved of symptoms by rest or added medications to dilute coronary arteries &

increase flow of adequate blood volume.

Sometimes these plaques can unfortunately rupture producing acute blockage to blood

flow starving heart of oxygen resulting in death of that part of the heart supplied by

particular branch of vessels. This is a medical emergency called acute coronary

syndrome, myocardial infarction or simply heart attack in lay terms.

Ref: Brief Overview of Coronary Artery Disease

http://heartdisease.about.com/cs/coronarydisease/a/CAD1.htm

30

3.6 Cancer

This is a conditions where cells in specific part or organ of the body begin to grow and

reproduce uncontrollably. These rapidly growing cells then invade and destroy

surrounding healthy parts of organs and tissues. The cancer cells begin in one part of the

body by progressively spread to other areas, a process called metastasis. The spread

occurs usually through continuity and by Lymphatic system as well as blood to much

distant areas. It is unfortunate that despite centuries of its known existence, of extensive

research, money spent on it and the amount of work put in, there is no solution to the

problem as we as yet do not know the exact cause for this abnormal change in growth

and formation of cancer cells in the first instance. It remains incurable to date and stamp

of death is synonymously associated with it by common man. However some factors

such as radiation, smoking etc. are known to be causative factors.

There are over 200 different types of cancers affecting body and some of the common

ones I have mentioned under the heading of Disease Prevalence and Incidence and thus

not repeated here. I should like to mention that cancer usually has an insidious onset and

in some cases it is quite advanced before coming to diagnosis. Hence if there are any

changes to body's normal processes such as bowel habits, occurrence of unexplained

bleeding or appearance of lump etc., then one needs to see his/her GP for consultation.

In early stages most cancers are curable in terms of 5 year survival rates. WHO records

show that cancer deaths reach the mark of 1.6million per year and every year millions

get diagnosed of it e.g. In UK alone in 2009 320467cases of cancer were diagnosed and

the commonest cancers found in UK affect Breast, Prostate, Lung, Bowel, Bladder and

Uterine (Cervical & Body) cancer.

Cancers of different organs and depending upon the stage at diagnosis apart from age

and sex, need different types of treatments. But generally speaking Chemotherapy,

Surgery, Radiation therapy form the main stay of treatment. Once again it is needless to

add that close follow up of patient is required in order not to miss recurrence.

Cancer - NHS Choices http://www.nhs.uk/conditions/cancer/Pages/Introduction.aspx

3.7 COAD (Chronic Obstructive Airway Disease)

As the name suggest it is a condition which results over period in obstruction to flow of

gases namely the exchange of Oxygen and carbon dioxide resulting chronic tissue

starvation for Oxygen and Accumulation of CO2 called carbon dioxide narcosis. This

condition usually affects middle and old ages and occur as a combination of Chronic

Bronchitis and Emphysema by constant irritation of Bronchi from irritants especially

tobacco nicotine; excessive secretion of mucus by airway cells lining and loss of ciliary

movement (fine hair like structures which line these cells and move to the mucous up).

There is also a loss of elasticity of lungs' sacs called alveoli.

31

The cumulative effect is collection of thick secretions, narrowing of passage and as

mentioned above obstruction to air flow and gaseous exchange. This conditions as said

above is particularly seen in Tobacco smokers which reduces their life expectancy

considerably Ref: Chronic obstructive pulmonary disease

http://www.nhs.uk/conditions/chronic-obstructive-pulmonary-

disease/Pages/Introduction.aspx.

4.1 Disease Prevalence and Incidence:

The prevalence of an illness or condition according to WHO definition "is the number of

individuals who have the condition at any moment in given time" WHO (2004)

http://www.who.int/healthinfo/global_burden_disease/2004_report_update/en/

Anaemia, hearing loss and migraine are the 3 most prevalent conditions in the world.

The other prevalent conditions such as arthritis, asthma, intestinal worms, visual

problems, major depressive episodes, injuries and cardiovascular diseases add to the list

with various levels of severity and multiple disabilities that cause health problems. The

GBD (Global Burden of Disease) links the average loss of health to disease and injury

but prevalence data does not indicate it.

Ref:

WHO (2004)


WHO The top 10 causes of death; http://www.who.int/mediacentre/factsheets/fs310/en/

WHO Burden of disease and cost-effectiveness estimates

http://www.who.int/water_sanitation_health/diseases/burden/en/

WHO About the Global Burden of Disease (GBD) project

http://www.who.int/healthinfo/global_burden_disease/about/en

Incidence and prevalence of chronic disease (MPKB)

Http: //mpkb.org/home/pathogenesis/epidemiology`

32

WHO (2004)http://www.who.int/healthinfo/global_burden_disease/2004_report_update/en/ Accessed on 30/5/2014

Table: 1: Prevalence (millions) of selected conditions by WHO region, 2004

The incidence of the condition is a number of new cases in a period of time - usually one

year. The diarrhoea disease is the most common cause of illness worldwide. The

incidence includes low, middle & high income countries. Pneumonia and other lower

respiratory tract infections are the 2nd

most common cause of illness globally. Other

illnesses such as Upper Respiratory tract infections (including common cold) and allergic

rhinitis (Hay fever) form the list.

Ref:

33

WHO (2004)


WHO The top 10 causes of death; http://www.who.int/mediacentre/factsheets/fs310/en/







The incidence of the condition "is the number of new cases in a period of time - usually 1

year" once again as per the WHO definition. The diarrhoeal disease is still the most

common cause of illness worldwide (indicating water - sanitation problem). The second

highest include lower respiratory tract conditions including Pneumonia and incidentally

includes low as well as middle and high income societies. Other infections such as upper

respiratory tract infections (including common cold) and hay fever add to the list

WHO(2004)

http://www.who.int/healthinfo/global_burden_disease/2004_report_update/en/.

34

Table: 2: Incidence (millions) of selected conditions by WHO region, 2004 WHO

(2004) http://www.who.int/healthinfo/global_burden_disease/2004_report_update/en/

Accessed on 30/5/2014

Cancer incidence is increasing and is relatively taking upper head globally. In 2004 11.4

million people were diagnosed in the whole world of cancer. Generally cancer incidence is

higher in high and middle income societies. Uterine cervical cancer is the only exception,

incidence of which is higher much more in African and South East Asian societies. Lung

cancer incidence is highest globally followed by Prostate, Breast, Colon and Rectum and

Stomach cancers. Prostate cancer affects more elderly population than younger males and it

is to be noted that the pattern of incidence of diseases and mortality and morbidity related to

35

They is changing world wide due to better health facilities. I mention here the list of Top 10

contenders of mortality (death|) between 2000 and 2012 worldwide to bring out this point.

i. Ischaemic Heart disease

ii. Stroke

iii. COPD

iv. Lower respiratory tract infections e.g. Pneumonia

v. Trachea Bronchus & Lung Cancers

vi. HIV AIDS

vii. Diarrhoeal diseases

viii. Diabetes Mellitus

ix. Road Injury

x. Hypertension

Figure 3: The 10 leading causes of death in the world; WHO (2014)

http://www.who.int/mediacentre/factsheets/fs310/en/ Accessed on 30/5/2014

WHO (2004)


36

WHO The top10 causes of death;

http://www.who.int/mediacentre/factsheets/fs310/en/







Figure 4: The 10 leading causes of death in the world by percentage; WHO

(2014) http://www.who.int/mediacentre/factsheets/fs310/en/ Accessed on 30/5/2014

Its starkly brings out the differences in 20th

and 21st century. Diarrhoea is much lower in rank

now though still in top 10 causes. HIV and AIDS have decreased but just slightly from 1.7

million to 1.5million. But chronic diseases particularly Diabetes is raising the head

worldwide.

37

It however makes most interesting reading to see the list of top 10 causes of death as tallies

were done in 1900 and 1997 ( almost century apart ) in USA. In 1900 in USA the top 10

were:

i. Tuberculosis

ii. Pneumonia

iii. Diarrhoea

iv. Heart Disease

v. Liver Disease

vi. Injuries

vii. Stroke

viii. Cancer

ix. Bronchitis

x. Diphtheria

In 1997 in USA top 10 tally shows a distinct change

i. Heart Disease

ii. Cancer

iii. Stroke

iv. Chronic Lung Disease

v. Unintentional injuries

vi. Pneumonia/Influenza

vii. Diabetes

viii. Suicide

ix. Chronic Kidney Disease

x. Chronic Liver Disease

38

Figure 5: The ten leading causes of death in the United States in 1900 and

1997; 2014 (MPKB) The Marshall Protocol Knowledge Base;

http://mpkb.org/home/pathogenesis/epidemiology Accessed on 30/5/2014

This is an interesting reading. One can see that acute infections like Diphtheria and

thank god chronic infections like Tuberculosis have disappeared from the list

altogether but unfortunately their place has been replaced by more chronic conditions

particularly Diabetes Mellitus which produces complications and adds to morbidity

and mortality. European pattern appears to be similar, cardiac, strokes, cancer and

chronic lung disease amongst the top 10 contenders. Ref:

WHO

(2004)http://www.who.int/healthinfo/global_burden_disease/2004_report_update/en/

WHOThetop10causes of death; http://www.who.int/mediacentre/factsheets/fs310/en/





Incidence and prevalence of chronic disease (MPKB) http:

//mpkb.org/home/pathogenesis/epidemiology`

39

Table: 3: Incidence (millions) of selected conditions by WHO region, 2004 WHO

(2004) http://www.who.int/healthinfo/global_burden_disease/2004_report_update/en/

Accessed on 30/5/2014

The current prevalence of chronic disease in USA throws some light. In 2000 approximately

125 million Americans (45% of population) had chronic conditions. 61million (21%) had

multiple chronic conditions. Even in children chronic health conditions in USA showed

distinct rise from 12.8% in 1994 to 26.6% in 2006 particularly for Asthma, Obesity,

Behavioural and Learning problems (JAMA 2010 - Ref)

Adults with the multiple chronic conditions are the major users of healthcare services at all

adult ages and account for more than 2/3 of healthcare spending (Ref).

40

A Dutch (European) analysis brings out the same message. The prevalence of chronic

diseases showed doubling between 1985 and 2005 with proportion of patients with 4 or more

chronic diseases increased by 300%. There is a further projected increase as also shown by

world report (Ref)

In 2002 leading chronic diseases, cardiovascular, cancer, chronic respiratory disease and

Diabetes caused 29 million death worldwide and annual mortality is expected to go up in real

numbers in relation to injuries and infections such as Polio, Rubella, and TB etc. WHO itself

described Diabetes as "world epidemic "with projected increase from 194 million to more

than 330 million by 2025. In fact CDC (Centres for Disease Control) projected to over 500

million in the next 25 years which appears more realistic realising the trend.

One can only imagine the health cost burden due to this and related co morbidities as well as

moderate to severe disabilities. A paper in 2010 by Boyle predicts that 1/3 of US population

will have Diabetes by 2050. It has also been predicted that by 2020 Atherosclerosis will be

the primary cause of death in the world, a truly shocking situation. Psychiatric disorders are

also on the increase and are expected to take over 1st place within the next few years. Once

again one has to imagine the cost burden of the conditions, their chronicity and problems and

stress levels in the society.

4.2 Burden of Disease and Cost Effectiveness Estimates:

As mentioned in above discussion poor sanitation and water quality still keep haunting us as

a threat to human health despite commendable work by WHO. The diarrhoeal disease has

much gone down in rank as a cause of mortality but it still accounts for estimated 4.1% of

TOTAL DALY (Disability Adjusted Life Year - use to quantify burden of disease from

mortality and morbidity ) and is responsible for deaths of 1.8 million annually as per the

estimate in 2004. Today in 2012 it is estimated at 1.5 million. Unfortunately Diabetes

mellitus has also shown a similar mortality of 1.5 million and increase worldwide. Naturally

expected increase in DALY. Nearly 88% of burden is attributable still to precarious and

unsafe water supply, hygiene and sanitation, and concentrated on children from developing

countries. (Ref)

WHO has already estimated the cost effectiveness of health benefits and other additional

benefits through better sanitation and safe portable water supply and other interventions for

each of the 14 sub regions it has divided the countries in two.

It is currently trying to estimate National level burdens of water related diseases and cost

benefits of safe water and sanitation services. Well, coincidently direct costs of Diabetes

Mellitus are estimated to be 9% of NHS budget and 14% of US Healthcare Budget (Type 2

Diabetes by Andrew Krentz & Bailey - 2nd

Edition).

This shows the urgent needs to put in equally more efforts in preventive, social and

management aspects of this disease Diabetes and hence my intention to concentrate in this

field.

41

4.3 Effectiveness of monitoring:

As the old adage in management goes "if you want to improve something, start by measuring

it or you won't see how to make a difference". In today's 21st century "world is a global

village" concept is aptly applicable, particularly because of computer science application and

fast modes of communication and transportation. The movement of people has increased

from one corner of the world to another and thus monitoring of health and disease has

become particularly important especially Infectious or communicable diseases and water

borne diseases and air pollution. WHO has been doing a commendable work in this field of

monitoring and particularly checking the effectiveness of these methods of monitoring

adopted e.g. international efforts of developing mass vaccination programmes in case of some

dreaded infections viral infectious disease, such as small pox, polio, influenza has led to

complete irradiation or marked reduction in the number of cases. Surge of small pox has

been eradicated from this earth and in India now Polio has been eradicated so has been in

developed world which is commendable.

Increasing improvement in water sanitation has resulted in Diarrhoea shifting its position

from amongst the first top ten killers (from 1st to 6

th position) as would be self-explanatory

from figures on previous pages. As also mentioned previously; monitoring list of top 10

contenders in USA in 1900 &1997 brings this out very starkly. Diphtheria has disappeared

from the list and place is taken by more chronic conditions especially Diabetes & Chronic

Lung Diseases e.g. COAD. Hence the importance of measuring effectiveness of monitoring,

so as also to fit in with changing incident pattern of diseases.

The truth about above statement is particularly brought out by the fact of more personalised

medicine trend in current age. With advances in Enzymatic Electrochemistry,

nanotechnology, physics etc.; there has been nearly an explosion in availability of cheap

enough gadgets, mostly portable in these days of consumer technology. As per the first

national survey on Self Tracking done by Pew Internet (2012) Ref: How self-monitoring is

transforming health - Medical News Today

http://wwwmedicalnewstoday.com/articles/264784.php 7 out of 10 adults Track one at least

one health indicator (60% track weight, diet or exercise and 33% track health symptoms e.g.

Blood Sugar, Blood pressure, sleep pattern, headaches, Heart rate etc.) and it is estimated that

in few years there would be nearly 170 million wireless gadgets around the world doing self-

health tracking. This trend in Self Health Tracking is "the biggest shake up in the history of

medicine" according to one of the leading Physicians Eric Topol.

The worry about the faulty monitoring either by gadget or an individual is being overcome by

technology developed by Professor Lionel Tarrasenko, at University of Oxford and Director

of Oxe Health in UK's Biomedical Engineering. I believe this clearly brings out the

importance of measuring effectiveness of monitoring if we are to tackle present day Top 10

killers on previous pages particularly Diabetes, Coronary Heart Disease, Strokes COAD etc.

42

Above parameters/Indices are very useful for health tracking or monitoring which should

now bring us to mentioning salient points of our this specific exercise in terms of aims &

objectives

5

5.1 Aims:

Our aim is to identify a Health Tracker Parameter & to develop a preliminary device to

monitor that parameter.

Having gone through the important common parameter/indices/markers I have discussed

above, I aim to select the parameter of blood glucose. In my further discussion I shall

mention the reasons for doing so, under the heading of Rationalisation. In this section I

mention it to clarify the thoughts. Similarly I should like to state that the device in mind to

monitor this parameter is a biosensor (Glucose monitor). This too is more discussed and

reasoned under the heading of Rationalisation and also in Biosensor.

5.2 Objectives

Identify parameters linked to well-being prioritise parameter list

Identify existing methods to monitor these

Design Prototype device

Test device & propose optimisation/further work

6

6.1 Monitoring Systems:

As mentioned above under the heading of effectiveness of monitoring an important old adage

in management that " if you want to improve something then start measuring or monitoring it

otherwise you won't see how to make a difference". I find this adage entirely appropriate and

thus re mention it. Latter half of last century and the beginning of current century has seen

considerable advances particularly in the fields of electrochemistry especially enzyme

chemistry as well as advances in physics (optical physics) in particular and in the field of

nanotechnology. The trend of having everything big is giving way to miniaturisation. The

things are thus becoming more cost effective and efficient leading to better measuring and

monitoring with the advent of age of sensors. A sensor is a device as the name suggest

senses or detects any change happening. There are usually 4 types of sensor systems in

common use currently:

1. Physiological sensors

43

2. Chemical sensors

3. Remote sensors

4. Biosensors

6.1.1 Physiological sensors

This is a system of sensors which help to measure or record some of the physiological indices

of stress, emotions etc. when a person is subjected to such exposures of psychological stress

and additive substances in natural environments and thus quantify them. Such a system of

sensors has been developed by team of scientists at Ohio State University arranged in the

form of wearable chest band. This provides steady measurements, with the use of two lead

ECG (Heart Rate and its pattern); Respiratory rate (with plethismography); skin/body

temperature (with surface probe thermistor), Skin conductance Response (SCR), motion

sensing using three axes accelerometer. All the sensors with their bio-amplifiers and signal

conditioning circuitry are assembled into two wireless motes connected to smart phones for

computer assessment Autosense: A Wireless Sensor System to Quantify Personal Exposures

to Psychosocial Stress and Addictive Substances in Natural Environments; Physiological

Sensors; https://sites.google.com/site/autosenseproject/research/chestband.

There is another connecting bridge mote which provides SD (Store Data) Card in case of

problems with smart phones or batteries to make the system fail safe about records.

Figure: 6 Mote 1 Form Factor; Sensor: ECG, Temperature, GSR, Accelerometer;

Autosense: A Wireless Sensor System to Quantify Personal Exposures to Psychosocial Stress

and Addictive Substances in Natural Environments;

https://sites.google.com/site/autosenseproject/research/chestband; Accessed on 17/5/2014

44

Figure: 7 Mote 2: Form Factor; Sensor: Respiratory Inductive Plethysmograph

(RIP); Autosense: A Wireless Sensor System to Quantify Personal Exposures to

Psychosocial Stress and Addictive Substances in Natural Environments;

https://sites.google.com/site/autosenseproject/research/chestband; Accessed on 17/5/2014

Figure 8: Bridge Mote Form Factor; Autosense: A Wireless Sensor System to Quantify

Personal Exposures to Psychosocial Stress and Addictive Substances in Natural

Environments; https://sites.google.com/site/autosenseproject/research/chestband; Accessed

on 17/5/2014

45

Figure 9 : End - to - End System; Autosense: A Wireless Sensor System to Quantify

Personal Exposures to Psychosocial Stress and Addictive Substances in Natural


on 17/5/2014

Figure 10: Signal captured from the Autosense system on a laptop appears below.

Red is respiration and green is the ECG; Autosense: A Wireless Sensor System to

Quantify Personal Exposures to Psychosocial Stress and Addictive Substances in Natural


on 17/5/2014

The signals from the system are visualised on laptop with continuous analysis of

Physiological changes for nearly a week at present. Such physiological sensors would be

quite useful especially in the field of medicine.

6.2 Chemical sensors:

A chemical sensor is a device that transforms chemical information (e.g.: composition of

substance under analysis, chemical activity or ions etc.) into a signal that can be analysed.

The information comes from physical property of analyte or by its chemical reaction. These

sensors usually have two components connected in series, a chemical molecule recognition

system called receptor, the other being a physicochemical Transducer. The receptor

interacts with molecules of analyte under investigation. This primary signal may be in

electrochemical, optical, mass, or thermal (heat) form. The transducer picks up the signal, it

is amplified by connected amplifiers and the final signal in the form of electrical current is

46

helpful to measure the concentration etc. (quantification) of system under investigation being

proportional. Chemical sensors have wide variety of applications such as Home Safety,

medical fields, Environmental (eg.to see level of pollution etc.) CHEMICAL SENSORS -

Nano-Bio Spectroscopy Group; nano-bio.ehu.es/files/chemical_sensors1.doc_definitivo.pdf

Figure 11: Composition of Chemical Sensor; CHEMICAL SENSORS - Nano-Bio

Spectroscopy Group; nano-bio.ehu.es/files/chemical_sensors1.doc_definitivo.pdf Accessed

on 17/5/2014

Examples of some of the chemical sensors seen in routine use are e.g. CO (Carbon

Monoxide) detectors, blood glucose detectors, Home Pregnancy tests kits etc. Just to

mention out of interest an insect (mosquito) possess variety of sensors in its antennas. One of

which is a chemical sensor which detects Carbon Dioxide (CO2) & Lactic acid produced in

breathe of mammals up to as long as a distance as 36 meters and this therefore results in

mosquito bites.

Nanotechnology has given a considerable boost to the use of chemical sensors in various

fields such as Communications, Transportation, medicine, safety and National security

CHEMICAL SENSORS - Nano-Bio Spectroscopy Group; nano-

bio.ehu.es/files/chemical_sensors1.doc_definitivo.pdf.

6.3 Remote Sensors:

These sensors gain information about objects or areas from a distance e.g. by means of

aircraft or satellites on which the sensors are fitted. The data collection is done with the help

of detection of energy reflected from earth, these sensors fall in 2 categories active or passive.

47

The passive sensors react to external stimuli. Recording the radiation reflected from the

earth's surface, normally from sun and hence can be used to collect the data only during the

daylight hours. Active sensors on the other hand use the internal stimuli to collect data about

earth e.g. by using laser beam to project by satellite etc. onto earth's surface and measure the

time it takes to reflect back to its sensor.

Remote sensing finds its applications in different range of fields including coastal, oceanic,

hazard assessment and Natural resource management such as wild life habitat &

environment.

Figure 12: A LIDAR (Light Detection and Ranging) image created with data collected

by NOAA's National Geodetic Service. March 20, 2014

http://oceanservice.noaa.gov/facts/remotesensing.html; Accessed on 17/5/2014

6.4

6.4.1 Biosensors:

A biosensor is an analytical device that incorporates a biological sensing element and

produces a specific response which can once again be converted with assembly of transducers

and amplifiers into a measurable current and thus quantify the concentration of analyte. With

progress in Electrochemistry and simultaneously in Nanotechnology the biosensors mostly

exploit the sensitivity and specificity of enzymes but other biological systems e.g. Antigen -

Antibody reaction, ligand binding and whole cell metabolism etc. are used.

http://oceanservice.noaa.gov/facts/remote-sensing.jpg

http://oceanservice.noaa.gov/facts/lidar.html

48

6.4.2 MARKET & APPLICATIONS:

The field of Biosensors is rapidly expanding, currently at the rate of 60% growth per year. It

has received major impetus coming from healthcare system or industry. E.g. approximately

6% of the western world population is diabetic and this number is rapidly growing all over

the world as discussed further under rationalisation. This population would benefit

immensely from availability of rapid, simple and accurate measurement of Blood Glucose

with a biosensor device which should be inexpensive, useable by common man. Hence the

expansion has had to be two pronged, one being such small portable glucometers for public

use as personalised medicine effectively decentralising the pathology laboratory which

otherwise uses high throughput expensive equipment.

Biosensors find wide variety of applications including in:

a) Medicine (mainly in glucose monitoring in Diabetes which is the major share).

Pharmaceuticals (drug discovery etc.),

b) other health related parameters measurement (e.g. cholesterol estimation)

c) Food and processing control (determination of drug residues such as growth

promoters etc.),

d) environmental monitoring( e.g. pollution, detection such as pesticides, heavy metal

contaminants of river waters),

e) defence and security(e.g. detection of airborne bacteria in bioterrorists activities)

The current estimated market is $13 Billion, much of (30%) is driven by Medical

Diagnostics particularly in Diabetes as mentioned already, the trend being towards

personalised medicine. The market currently utilising only 0.1% capacity with a

huge scope for expansion. (Ref Biosensors: sense and sensibility; Anthony P. F.

Turner; Chem. Soc. Rev, 2013; 42, Pages 3175-3648.)(Enzyme Technology; what are

Biosensors? http://www1.1sbu.ac.uk/water/enztech/biosensors.html)

6.4.3 The Basic Components of Biosensors:

1. Biological recognition element

2. Transducer

3. Amplifier

4. Microprocessor with display unit

49

Figure13 : Schematic diagram showing the main components of a biosensor. The

biocatalyst (a) converts the substrate to product. This reaction is determined by the transducer

(b) which converts it to an electrical signal. The output from the transducer is amplified (c),

processed (d) and displayed (e). Enzyme Technology (2012) what are Biosensors?

http://www1.lsbu.ac.uk/water/enztech/biosensors.html; Accessed on 17/5/2014

Figure 14 :Working principles of a biosensor

Figure 15 : Glucose Detector

50

6.4.4 Requirements of Biosensors

A successful biosensor needs to possess certain beneficial features. To enumerate:

i. It should have demand ( this can be met as seen above)

ii. It should have a Biocatalyst under normal conditions of storage and be specific

iii. As far as manageable, the reaction produced in the sensor analysis should be

independent of physical parameters such as temperature etc.

iv. In case of invasive monitoring it should not be prone to fouling and be very tiny, non-

antigenic and sterilisable.

v. The response needs to be precise, accurate and reproducible

vi. Should have a good portability, cost effectiveness (cheap) and useable by common

person

(Enzyme Technology; what are Biosensors?

http://www1.1sbu.ac.uk/water/enztech/biosensors.html)

The biological recognition element is also called bio receptor and can be in many forms but

the commonest bio receptor use:

a) Enzymes - though they are costly, are highly selective and specific and above all

sensitive in their action. As enzymes do not get used or destroyed in the reaction but

only catalyse it, it is reusable many times before it loses its sensitivity. The problem

is with stability. However reusability as well as reproducibility of results makes it

affordable and attractive element. Other bio receptors used are:

b) Cells - they remain attached to the surface, remain active for a long time, and are

reproducible and reusable.

c) Antigen - antibody reaction - specific binding capacity of antibodies to certain

corresponding antigen is made use of in this form of biosensor. The binding event is

recognised with the use of tracing elements such as enzymes or fluorescent molecules

which produces a signal. The sensor is immunosensor.

d) Organelles - these are independent functioning units of cells with their enzymes to

perform those functions. Commonly used organelles include chloroplasts,

mitochondria (used for detection of calcium concentration of given medium) and

lysozymes.

51

6.4.5 Transducer/ Bio transducer:

This is a highly important unit of biosensor. It is the physicochemical change

recogniser and transductor (convertor) of biological signal produced (not quantifiable)

into a quantifiable electric current or voltage (in potentiometers). Biological

recognition element and Bio transducers are the biological components of biosensors.

The remaining three namely amplifier, microprocessor and display unit are electrical

components of the sensor and mostly are assembled together and many a time a more

costlier part of the system.

6.4.6 Functioning of Biosensor:

The biological recognition element reacts with the specific analyte that an analyst is

interested in. The biological signal produced by the reaction is picked up and

converted into quantifiable unit namely the electric current. The generated current is

amplified by amplifier with the suppression of noise or interference. Processed by

microprocessor this analogue signal is converted to digital signal finally to be

displayed by the display unit, as concentration of analyte as the produced electrical

current is proportional to the concentration (Enzyme Technology; what are

Biosensors? http://www1.1sbu.ac.uk/water/enztech/biosensors.html).

6.4.7 Classification of Biosensors:

The biosensors can be classified based on its transduction or detection element or

alternatively as per by its biological recognition element.

Classification by detection/ transduction:

This would include 3 electrochemical methods namely:

Amperometric (using movement of electrons produced in redox

reaction)

Potentiometric (changes in the distribution of charges producing

electric potential)

Conductometric

Others such as Optical, Acoustic and colorimetric (measuring heat

output of the reaction). With advances of optical physics the optical

52

sensor systems may be based on surface Plasmon response (SPR) or

merely changes of absorbance or fluorescence.

Piezoelectric (where effects due to mass of reactants or products is

measured). Piezoelectric element is incorporated in the biosensor in

the form of crystal and binding of analyte changes the resonance

frequency which gives binding signal.

Classification by Biological Recognition Element:

Classification on this basis can be further subdivided into 2 categories:

a) Bio catalytic recognition

b) Bio affinity recognition

a) Bio catalytic recognition - in this mechanism of sensors either an enzyme, a whole

cell, cell organelles or biomimetic material or tissue as discussed above are

immobilized on the transducer as a biological compound or recognition element.

b) Bio affinity recognition - the recognition element is DNA and RNA based or antigen

antibody based ( immunosensors - see above) as well as can be receptor/agonists/

antagonist biosensor using membrane and capture configuration

DNA/RNA methods employ nucleic acids interactions recognition is based on principle

of complimentary base pairing and the sensors are called genosensors.

Developments in Nanotechnology has boosted the field of Biosensors with

measurements to molecular and atomic levels.

6.5 Glucose Biosensors:

Having discussed Biosensors it is now appropriate to discuss glucose Biosensors in

particular, since in my aims and objectives I have clearly mentioned my choice is to track a

health tracker parameter - blood glucose.

Glucose Biosensors though well-known and established as important health tracker parameter

in diabetics for testing as point of care (POC) or home and personalised medicine, also has

many other biotechnological, food & beverages, pharmaceutical industrial as well as

53

chemical industrial applications(Banker et al; 2009). It makes one understand their

importance

6.5.1 A brief historical perspective of glucose sensing

During mediaeval times attempts were made to diagnose diseases by examination of urine for

appearance, colour, sediment and even taste. In early 19th century glucose was identified in

urine as the sugar present in Diabetics later corroborated in 1838 by George Rees, from Guys

Hospital in London who isolated excess sugar in blood OD Diabetics. Stanley Benedict

devised a test in 1908 for urinary monitoring of sugar which was used for next 50 years 1970

- 1980s were years Anton Clemens at Ames developed quantitative blood glucose meters

developed by Life scan in 1980 in UK (1980 - 90) saw miniaturisation of glucose meters.

The choice slowly changed to Biosensor with enzyme chemistry with advances in

Electrochemistry with advances in Electrochemistry. The first glucose Biosensor system

ExacTech was launched by Medi sense in 1987 using enzyme electrode strips developed in

UK by Cranfield University & Oxford University (History Committee, IBMS, London;

March 2012). 1990 - 2000 saw increase in production of enzyme technology based glucose

biosensors as well as ISF continuous glucose monitoring. Abbott Labs, Bayer, Roche &

Life scan were the 4 companies dominating in the production of glucose meters totally

capturing 80% of world market. By 2000 the Reflectance technology based glucose meters

had become obsolete. (Rich Mendoza 1999).

6.5.2 Generations of Biosensors

Over the past 40 years various advances have taken place in the techniques and designs of

glucose sensors production. Depending on this the sensors could be divided into 3

generations but in order to realise the monitoring of diabetes control one needs to understand

how bio electrodes function. The figure below shows the working principle of Biosensors

along with figure above. The bio electrodes mostly use enzymes, the commonest being

glucose oxidase (GOx). The first glucose electrode used enzyme GOx entrapped over

oxygen electrode using a semi permeable dialysis membrane. This is the first bio electrode

and all biosensors are based on this original glucose enzyme electrode. The measurements

based here were based on O2 consumption by enzyme catalytic reaction

Glucose + Oxygen →GOx Gluconic acid + H2O2

Since then attempts in various forms have been made to improve electron transfer from active

site of Go to the electrode and to reduce the transfer distance.

54

6.5.3 First Generation Glucose Biosensors

This generation Biosensors relied on use of natural O2 cosubstrates production and detection

of H2O2. The electrons are transferred here from glucose to electrode in 2 steps:

Step 1: Reduction in Flavin group in the enzyme (FAD) to reduced form of enzyme (FADH2)

GOx (FAD) +Glucose →GOx (FADH2) +Gluconic Lactone

Step 2: re oxidation of Flavin by oxygen to give back re oxidised form of enzyme GOx

(FADH2) +O2 →GOx (FAD) +H2O2

Here the measurement of H2O2 produced are done using Pt (Platinum) electrode and

entrapped GOx between inner cellulose Acetate membrane internally (for anti-interference)

and outer biocompatible membrane. The problem with this generation of sensors is

limitation of oxygen diffusion to enzyme.

6.5.4 2nd Generation Glucose Biosensors

These 2nd generation sensors made use of mediators (non physiological synthetic) as electron

acceptors from redox centre of enzyme to electrode. Enzyme wiring was used to improve

the contact and also to get over problem of oxygen diffusion by use of mediators.

This involves direct transfer of electrons from glucose or analyte to electrode via active site

of enzyme with the total elimination of mediators. This therefore gives rise to high

selectivity (Malhotra & Turner 2003).

6.6

6.6.1 Problems to surmount in Glucose Biosensors

There are two main problems in the Glucose Biosensors which need to be carefully

overcome, one is to get correct reliable blood glucose readings, on which the dosage of

medication in the patient depends and there have been instances in the past with false

readings leading to wrong dosages of insulin heading to death. Hence importance of

reliability. The problems are mainly the two interference and fouling. Interferences can be

enzyme interferences or electrode interferences.

55

6.6.2 Enzyme Interferences

As mentioned in the 2nd generation Glucose sensors many manufacturers used glucose

dehydrogenase (GDH) instead of good old glucose oxidase (GOx). This obviously needed

cofactors such as FAD (Flavin Adenine Dinucleotide); PQQ (Pyrroloquinone Quinoline - see

reaction under 2nd generation sensors and NAD (Nicotine Adenine Dinucleotide). However

due to problem of instability apart from main concern of non-selectivity with cross linkage to

other sugars such as maltose, galactose and Xylase producing interference and unreliability

of results, the manufacturers had to stop GDH - PQQ and switch over to GDH - FAD

combination. Hence these mediators based 2nd generation Biosensors became unpopular.

GDH - NAD combination was however unaffected. There were however tragically more

than 100 deaths reported between 1992 and 2009 related to faulty glucose monitoring due to

various causes (Newman 2010).

6.6.3 Electrode Interference

Most glucose biosensors incorporate bio electrodes. Usually there are 2 or 3 types of

electrodes employed, namely working electrode, reference electrode and counter electrode.

Unfortunately all ox disable substance or species can get oxidised at the electrode without

selection including glucose but also substances like vitamin C (Ascorbic Acid),

Paracetamol(Acetaminophen) or uric acid which are also commonly present in blood apart

from glucose and thus may interfere with estimation of glucose concentration(Piechotta et.al

2005). Many efforts have gone into removing these interferences by coating of electrodes

with polymer membrane or mediators or some pre oxidising membranes meant for

interferents to make them non electro active. But nothing has worked satisfactorily. On the

contrary the membranes suppressed glucose signals and affected measurement. Attempts

therefore were made to use ISF (interstitial fluid) with a view that concentration being low,

there may not be appreciable interference. Other electrophoretic separation methods also

have been tried but the matters have got more complicated, more error from and also more

expensive. The research is continuing (Wang 2002).

56

6.6.4 Fouling

As stated above this was the serious problem to overcome. In vivo applications in particular

caused this problem of fouling of electrodes by various species such as whole cells and

proteins. New materials which would mimic the properties were developed to improve the

function. The modifications of polymers used in biosensors though a good way to get over

the problem, has not been satisfactory yet lining of electrodes with electro polymerised

conducting polymers has held some promise in overcoming this problem. Finally attempts

have also been made with use of anticoagulants notably heparin onto surfaces but this has

been ineffective and caused leaching into sample solutions (Zhang et.al 2000).

It would be appropriate at this stage to discuss the various ways currently being used to

monitor glucose in the body followed by more detailed discussion of enzymes as it is my aim

and objective to develop a device to measure blood glucose using enzymatic application in

this sensor, before we proceed to the next chapter of materials and methods.

There are 3 ways correctly being undertaken in Glucose measurement.

6.7.1 Measurement by finger pricking (Point of Care)

This is the single one off measurement of blood glucose by minimally invasive but painful

traditional method of checking the blood sugar at any given time in order to adjust the dosage

of medication (Insulin in particular). This is generally effective if done regularly in

conjunction with regular check-up of HbA1c to see the glycaemic control in previous three

months and suffices if the diabetic control is really good. However it does not give idea

about ups and downs in blood on continuous basis in order to avert the problems of hyper or

hypoglycaemic fluctuations over 24 hours and been painful every time to take the prick to a

diabetic.

All attempts have been made to split only the superficial layers of epidermal layers of skin

and to collect only interstitial fluid (ISF) for measurement to avoid pain but, are not

successful and not reliable either till to date.

6.7.2 Continuous Measurement of Glucose:

In the last 15 years especially in last 10 years attempts have been made putting in

considerable efforts and money to develop a minimally invasive subcutaneous insertion of

biosensor to get continuous monitoring of glucose levels in ISF which almost represents

matching blood glucose levels.

57

However there are times of rapid fluctuations in levels especially hypo state when blood and

ISF glucose levels may not match.

This has been the main obstacle in the development of close loop miniaturised implantable

computer system for delivery of regular as required dosage of insulin to maintain blood

glucose levels to near normal all through the day ( the so called artificial pancreas). Apart

from this the problems of fouling of electrodes and interference as stated above are still not

mastered. Hence the lag (Wilkins & Atanasov 1996).

6.7.3 Non-invasive Glucose Measurements by sensors

This has been a great idea difficult to implement. Non Invasive measures as the name

suggests do not involve mechanical invasion of body to collect the blood. But the

information about sugar levels is obtained either by sophisticated methods of advanced

science of Physics or by other body fluids namely secretions e.g. urine, saliva, tears or even

sweat. Regards these secretions lots of factors affective for e.g. exercise, diet or outside

atmospheric temperature in case of sweat. Secondly these secretions may not exactly match

their sugars corresponding to blood levels and with urine there may not be necessarily any

sugar to measure in case of hypo or euglyceamia and in established diabetics.

In case of use of science of physics e.g. Raman Spectroscopy, Ultrasound, Electromagnetic

and Fluorescence techniques, Calorimetry, Polarimetry etc. The measurements so far have

not been satisfactory especially in case of hypoglycaemic episodes and methods expensive.

Hence no further advancements compared to standard invasive blood glucose measurements

(Wilkins & Atanasov 1996).

6.8.1 Enzymes and their mechanisms of action

An enzyme is a macromolecule of protein in its simpler form made of large polypeptide

which helps to speed up the process of catalysis of specific substrate into product, but does

not itself suffer the chemical change. Hence this property makes it reusable and affordable as

enzymes are generally expensive as well as have a drawback of instability.

The current exercise in this work involves construction of a measuring device for health

tracker parameter of blood glucose.

I wish to construct biosensor with use of enzymatic reaction for this. Normally the common

enzymes used for glucose are GOx (Glucose Oxidase) and GDH (Glucose Dehydrogenase)

but GDH has a problem of NAD cosubstrate needing mediators. I wish to use Glucose

Oxidase which is also commercially produced on a large scale and been reasonably

affordable.

58

One of the ways to use given enzyme repeatedly is to immobilise it. The enzyme is a soluble

biomolecule. If we attach this to some solid phase it will become insoluble and attach there.

The process is called immobilisation and therefore as said reusable enzyme.

6.8.2 Methods of Immobilisation of Enzymes (with Advantages and disadvantages)

Figure 16 : Principles of different enzyme immobilisation methods (adapted from Turner et

al., 1987)

6.8.3: Physical Adsorption

This is the oldest and simplest inexpensive method and useful on cellulose support materials

(Turner et.al 1987, Malhotra & Turner 2003). It makes use of weak binding forces such as

Hydrogen Bonding & Van der Val. But as the forces are weak, the enzyme remains unstable

change of pH & temperature makes it more susceptible to desorption.

6.8.4 Enzyme Entrapment

In this process the immobilisation is done by trapping of enzyme molecules within the matrix

pores need to be sufficiently large to accept enzyme molecule to enter and ideally no other

species so as to avoid interference(Turner et.al 1987). The method is in common use these

days but the problem is with denaturation and enzyme escape if matrix molecules are too

large.

`

59

6.8.5 Micro Encapsulation

In this method the enzyme is immobilised in an artificial membrane. The product entity

crosses the membrane by diffusion process. The large molecules of enzyme are within this

synthetic membrane. The advantage of this method is it is good for large macromolecules of

enzymes and large surface area per enzyme than in entrapment. This method however has

not found much favour in biosensor mechanism (Malhotra & Turner 2003).

6.8.6 Cross Linking

The reagents in this method are Bifunctional and the method can be used in combination with

entrapment or other method. The most common commercial use is with gluteraldehyde.

Unfortunately the enzyme can cross link itself to reduce its power of activity.

6.8.7 Covalent Bonding

In this method covalent bonding occurs between the enzyme and membrane matrix or to the

surface of support material. The enzyme activity gets protected of immobilisation is done in

the presence of substrate (Malhotra & Turner 2003 & Turner et.al 1987). It is most stable

form of immobilisation as binding forces or bonding is powerful covalent bonds. However it

is a complicated process and also thus expensive. The choice of immobilisation method

depends on the physical properties of analyte also in conjunction with the type of support

material. The most common methods in use today are adsorption and covalent bonding

(Malhotra &Turner 2003).

6.9 Various uses of Enzyme (Glucose Oxidase GOx)

Glucose Oxidase has many industrial applications and thus is produced in commercial

quantities making it relatively cheap.

Apart from use in enzyme based (bio electrodes) in Glucose sensors for management of

Diabetes (more than billion strips/year), the enzyme is also used for food and beverage

industry. It is also used as colour and flavour additive for stabilisation as well as additive to

extend the shelf life of products. It is used also in fermentation industry to determine the

Glucose levels. It now finds its use in biofuel cells for the production of electrical energy

from biochemical energy (Banker et.al 2009). Finally it is also used in textile industry for

bleaching, for oral hygiene and antimicrobial products as well as bactericides.

60

I should like to mention here the mechanism of action of GOx.

Oxidation of glucose is an important process explained in 2 stages:

Stage 1: in the presence of molecular O2

Glucose+O2 ↔Glucono - ᵟ - Lactone + H2O2

Stage 2 Spontaneous hydrolisation of Glucono - ᵟ - Lactone

Glucono - ᵟ - Lactone + H2O2 ↔ Gluconic acid

(Hence final outcome)

Glucose+ O2 →Gluconic acid+ H2O2 (this is measured in sensors)

61

CHAPTER 2: MATERIALS & METHODS

1.1 Materials & Methods (for Electrochemical Biosensors)

In this chapter I first wish to discuss and review the various materials and methods currently

available in the field and then revert to my intended choices of materials and method

proposed Materials used in electrochemical biosensors can best be considered under 4

heading:

i. Biological recognition elements

ii. Materials for electrodes and supporting substrates

iii. Materials for fabrication of outer membrane

iv. Materials for immobilisation of biological recognition elements

This will lead us into methods currently available in general and then naturally proposed

method for experimentation and construction of the device (Biosensor for Glucose

Monitoring)

1.2 Biological Recognition Elements:

These elements can be enzymes, antigens, antibodies, mediators and cofactors. Amongst the

enzymes used the most common enzymes is Glucose Oxidase (GOx) as already mentioned

and having its various applications and mode of reaction described under section (1.). I

should like to once again bring out the point the point that is available in commercially

produced quantities and thus relatively cheap than others. Hence my choice would be for this

enzyme in order to be cost efficient without sacrificing the quality of sensor. Incidentally

this was the enzyme used in first electrochemical biosensor introduced by pioneering work of

Clark & Lyons 1962. The only difference it measured then the oxygen consumption using

oxygen electrode of Clark whereas at present in our methods we measure H2O2 generated.

The other enzymes used commonly are Lactate Oxidase (Vadgama et.al 1986) and lactic

dehydrogenase (LDH) but this requires use of mediators for efficiency improvement. These

enzymes make use of principle of Bio catalysis. The Bio affinity sensor such as

immunosensors use antigen - antibody reaction. The others being genosensors which use

principle of nucleic acid Hybridization with DNA base pairing. In the last decade using

electrochemical, optical & piezoelectric transducers. Much work has been done on these

DNA (genosensors), Biosensors e.g. PNAs (Peptide nucleic acids ) have effective

hybridization properties and have been found to have considerable more advantages in

sequence specific recognition with higher sensitivity and specificity and faster hybridization

at room temperature.

2.1 Material for Electrodes and Supporting Substrates:

An electrode used in these biosensors is essentially a transducer that recognises a biological

non quantifiable signal of reaction and helps to Trans duct it or convert it into a quantifiable

electrical or any other physicochemical signal. There are usually 2-3 types of electrodes in

62

use as stated above. Working electrode where electrons from Redox centre of enzyme are

usually transferred. Various metals such as Platinum (Pt), Gold (Au), Silver (Ag), &

Stainless Steel have been used for the preparation of solid electrodes due to their excellent

electrical and mechanical properties and conductivity. Apart from the recent years have seen

use of carbon material e.g. Graphite, Carbon Fibre & Carbon black for being chemically inert

but with pure crystal structure and high signal to noise ratio. There has also been use of

composite materials made from two dissimilar material with different properties which are

retained but the composite has very distinct physical, chemical & mechanical properties

different from original material e.g. carbon polymers composite. The enzymes used as

biological recognition elements can be immobilised on these electrodes. In this case organic

polymer have derived considerable attention as the materials provide H2O2 oxidation current

at a very low potential than other sensors.

2.2 Membrane Materials:

The covering thin membrane for biosensor electrodes helps to perform several functions such

as mechanical protection of this sensing probe, reduction of interference, diffusion control

etc. There are commercially available polymers such as Polymethyacrylate, Polyurethane,

and Polyvinylchloride (PVC) etc. which are commonly used in Biosensors used in

environmental monitoring or food production but when the sensor used finds contact with

Biological medium it can affect the functional stability due to biocompatibility problem. This

is particularly important in glucose biosensors. Apart from this above mentioned function of

reduction of interference was specially studied at our Cranfield University (Reference:

Catalytic Materials, Membranes and Fabrication Technologies for Construction of

Amperometric Biosensors by J.D Newman.et.al; Anal. Chem 1995, 67; Pages 4594 - 99).

The study revealed that apart from cellulose acetate the best material for reduction of

interference was Nafion (Perfluorosulphonic Ion Exchange Membrane) which produced

higher currents and to prevent fouling of electrodes by Proteins and Platelets. Polyethylene

Glycol (PEG) is also extensively used to modify the surfaces. In my proposed method I wish

to try to use these for problem solving.

2.3 Materials for immobilisation of Biological Recognition Elements:

In section (1.) I have already mentioned about the enzyme immobilisation ways where

crosslinking, entrapment etc. have been discussed more. Hence therefore would suffice to

mention some materials commonly used in this process of immobilisation. Commonly used

materials are Gluteraldehyde and Hexamethyldiisocynate. These work by forming crosslinks

between Bio catalytic species or proteins shown in figure under section (1 :). This process is

also known as reticulation. Other methods including entrapment can be done using

nonconductive polymers like polyacrylamide and polyphenol. Organic polymers are also

used for the purpose such as carbon conductors. These are deposited electrolytic ally onto

conducting support for immobilised enzymes, the reaction causes the transfer of electrons to

Redox Polymer gel which are in entrapment process of enzymes. Latex particles can also be

used as substrate for covalent bonding of enzymes to immobilize them.

63

I shall now proceed to various methods available for the construction of Biosensors as

materials, fabrication technology and designs are all intricately related.

3.1 Methods for Fabrication:

Most commonly available sensors in the market make use of either Amperometry or

Potentiometry in transduction by Transducers which is the essential element of quantification

of signal (see classification of Biosensors). In personal glucometers Amperometry is more

commonly used and I intend to use the same techniques namely construction of my

amperometric enzyme electrode. Hence a little more about amperometry and potentiometry

would be pertinent here before discussing available methods.

In Amperometric Transduction the potential is kept constant with respect to Reference

electrode. The Reference electrode of Ag/AgCl (electrically stable). The current generated is

measured after the oxidation or reduction of electro active species on the surface of working

electrode. Small variations do not alter the rate of process at surface of working electrode.

The generated signal however is dependent on transfer of electro active species to electrode

surface causing problems of interference and fouling diminishing its sensitivity. This is the

drawback of amperometry which makes it as yet not so suitable for continuous monitoring.

In my biosensor I intend to work of these problems of electrode as mentioned.

In Potentiometry on the other hand the transducers measure the difference in potential

between the active (working electrode) and Reference electrode needs to be very stable.

There are incidentally 3 types of such electrodes in use:

a. Ion selective electrode (ISEs)

b. Gas Sensing Electrodes

c. Field Effective Transistors (FET) which are more difficult conditions to handle.

Hence Amperometry is preferred. Other techniques of transduction are mentioned

under their classifications.

3.2 Electrode Fabrication Methods in current use:

The techniques used in fabrication of conductive supporting substrate made of gold, platinum

or carbon rod or paste, polymer etc. are generally classifiable into:

i. Screen printing

64

ii. Deposition

iii. Polymerisation

iv. Plasma induced polymerisation

v. Photolithography

vi. Nanotechnology

3.2.1 Screen Printing:

This is a thick film - method used in industry for mass production. The technique involves

printing paste material onto the matrix directly using mask net with designed pattern on it.

Carbon or graphite screen printing is routinely used for production of electrodes for

Biosensors. Recent improvements have seen use of epoxy based polymer. Screen printing

has previously been used at Cranfield University Labs. (Reference: Catalytic Materials,

Membranes and Fabrication Technologies for Construction of Amperometric Biosensors by

J.D Newman.et.al; Anal. Chem 1995, 67; Pages 4594 - 99). Hence availability is reasonably

assured particular. I intend to use epoxy + carbon composite in my procedure. Even enzyme

glucose oxidase intended for use can be mixed in the paste for printing to produce electrodes

and technique is particularly suitable for electrode arrays to analyse. Blood, serum

electrolytes & metabolites simultaneously: a similar close technique is ink jet printing.

3.2.2 Deposition Method:

By this technique which is thick/thin film based; can be used in Biosensors to produce an

electro conductive film deposition on the substrate for electrode. The deposited film can be a

metal complex or metal. The biological element too can be deposited in the same go.

3.2.3 Polymerisation:

Use of heat or light or electrochemical is made to rearrange bonds within monomer to make a

polymer out of it. Electrical conductivity property can be induced by use of metal powder

into monomer before the process of polymerisation. This technique needs a use of only a

small potential of up to 200mV only. The transfer of electrons is facilitated especially if

enzymes immobilised by entrapment are used.

3.2.4 Plasma Induced Polymerisation:

This is a highly specialised procedure to carry out. Polymerisation in high vacuum.

Polymerisation gas plasma is coated onto the substrate surface which helps to produce a very

thin coating chemically and mechanically stable. It helps to introduce a large amount of

Biological material onto the surface.

65

3.2.5 Photolithography:

This method is in use for a long time in semiconductor to produce integrated chips. Light

passed through a photo mask is cast on photo degraded material surface to get pattern and the

technique is useful to fabricate micro - lens arrays for sensors.

3.2.6 Nanotechnology:

This technology is used in miniaturisation, micro fabrication and in production of immune

and geno sensors. Atomic Force Microscopy (AFM) is used in this method.

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CHAPTER 3: PROPOSED METHOD & RATIONALISATION

1.1 My proposed Method for the Glucose measuring Device and Schematic plan

(What can we do better?):

Measurements of Blood Glucose levels has following basic requirements to be considered:

i. Response of measurement needs to be fast :(within 1-5minutes)

ii. It needs to be accurate: this is the most important as on this the management of

Diabetes depends. This means inaccuracy due Interference species and Fouling of

electrodes have to be overcome. Maximum deviation permitted (˂10mg/dl).

iii. It must be sensitive: the signal to noise ratio here must be high. Thus detectable

signal should be as small as 0.1 mM or approximately 2mg/dl change in

concentration.

iv. Range: Normo glycaemia is not much difficult to assess but it is the Hypo or

Hyperglyceamia that matters to be reflected particularly Type 1 diabetics. Hence

fluctuation Range detectable should be from 1 - 30mM or 20 - 600 mg/dl to control

most eventualities.

v. The measuring device should be stable - the glucose signal should not deviate more

than ± 5%during the lifetime of device (Reference: E. Wilkins & P.Atanasov; Review

Article; Med. Eng. Phys; Vol 18; 1996).

Hence following steps in preparation

1.2 Fabrication of Electrode (working electrode):

a) As per previous studies done at Cranfield University – Jeff D Newman et.al;

Anal.Chem.1995) Graphite Powdered (MCA Services; Royston; UK) → MCA4

added to disperse in Epoxy (Polymer resize).

b) Pre immobilized glucose oxidase on carbon particles by adsorption mechanism. Now

to be mixed with (a) to form an integrated working electrode - left to dry for 2 - 3 hrs.

If many electrodes proposed for array (screen printing using DEK 247) DEK Printing

Machines, Weymouth, UK could be tried provided the composite material remains in

semisolid paste stage for sufficient time. (The total quantity and proportion of

Graphite Powder as well immobilised enzyme to be worked out.)

Reference Electrode: - to be made as saturated aqueous electrode made of Ag/AgCl

→proportion to be worked out.

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1.3 Construction of outer membrane:

This as discussed previously essentially will help to protect working electrode prone

problems of fouling & Interference also controlling diffusion at the same time. This

membrane has to be a double coating (I) Nafion (perfluorosulfonic ion exchange material)

followed by coating of Polyethylene Glycol (PEG) →This should reduce interference,

producing high current and prevent fouling.

Finally The voltage required is reduced to 300 - 400 V sufficient as MCA4 material is used.

Connect directly to Assembly Amplifier & microprocessor with display unit (Electrical

Component).

The justification of this technique is further discussed under Rationalisation.

2.1 RATIONALISATION

In the discussion that follows, I have decided to concentrate and focus my attention to

Diabetes Mellitus and to devote my work to the development of measurement of Blood

Glucose which I consider after careful thought, one of the most important parameters of

health tracking systems. In the 1st part of the discussion I shall attempt to explain why I have

chosen Diabetes Mellitus of all the diseases. In the 2nd

part of the discussion I shall try to

explain the importance of measuring the Blood Glucose regularly and by the patient himself.

In the final part I will try to explain the reasoning behind my proposed method for the

development of the device - the Glucometer.

The preference to this condition as already mentioned in previous discussion Diabetes

Mellitus is a chronic condition of Hyperglycaemia resulting from either defect in insulin

condition secretion or insulin action or both in combination. The sustained hyperglycaemia

results in complications of macrovasculature and microvasculature as well as nerves. This

causes a prolonged morbidity and premature mortality - Diabetes is known to reduce the

normal lifespan expectancy of a person on an average of 10 years and it affects obviously the

individual's well-being and thus the fruitful contribution to society, nation and world at large.

(Type 2 Diabetes in Practice; 2nd

Edition by Andrew J Krentz & Clifford J Bailey;

Southampton University Hospitals UK).

The Macro vascular complications resulting from associated atherosclerosis produces

conditions of coronary Heart Disease (CHD) and Strokes Microvascular complications

produce nephropathy and renal failure, Diabetic neuropathy and retinopathy leading to

blindness in uncontrolled cases. All these add to the morbidity and premature mortality

reducing the productivity of an individual and his contribution to the society. Unfortunately

during his working lifespan it is needless to add its effects on the life of family members.

Even in the current situation where many of the factors in the production of diabetes and its

management are not known; one thing is certainly known, and that is if we can manage the

68

blood sugar control to near normal; by constant Blood Glucose and cholesterol, monitoring,

adjust the life style of person regards exercise, diet, weight reduction & prevention of

smoking, which are simple and manageable procedures, then the diabetics can lead almost

normal healthy & full life span. This is one of the reasons for my particular interest in this

condition.

As per the expression of WHO itself the incidence of Diabetics all over the world is

increasing rapidly "like an epidemic". In 1985 estimated 30 million people worldwide had

Diabetes. By 1995 number was135 m by 2000 - 177million. In 2003 the estimate was 194

million. It was expected to reach 330 million by 2025. But as per the estimate of CDC

(Centres for Disease Control) it was likely to touch nearly 500 million. The burden is more

on relatively less developed countries. The increase in India was expected from 38million to

73 million and China from 22million to 46 million. These two are the most populous

countries of the world; others like Pakistan, Indonesia and Mexico follow along with the

other less developed world. Incidence is nearly 3-4% in European countries & 7% in USA

and in USA one in 3 persons born in 2000 are expected to develop diabetes sometime in their

lifetime (Type 2 Diabetes in Practice; 2nd

Edition by Andrew J Krentz & Clifford J Bailey).

The figures are simply colossus and mind boggling and hence if I can be of any use in

helping these individuals in maintaining their lifespan to normality and thus indirectly help

the life of their family well-being and happiness, then every effort is worthwhile. Hence my

interest in this condition of chronic disorder of Diabetes Mellitus.

As I have brought out in the previous discussion been the two tallies done in USA in 1900 &

1997 (almost a gap of century) the top 10 contenders as harbinger of death & morbidity the

place of acute infections such as diphtheria (this has disappeared from the list ) has been

taken over by Diabetes. Even worldwide it is the (8th)

and in the western societies the

5th

cause of mortality. As per previous WHO estimates number of deaths attributable to

diabetes was just over 800000. Currently 1.5 million but of top 2 contenders of coronary

heart disease and strokes who find their causation in Diabetes & atherosclerosis are to be

considered, then according to WHO more plausible figure to be likely is around 4million

deaths per year related to Diabetes i.e. around 9% of global total. One needs to concentrate

on this chronic disease in particular if we are to achieve the national and global wellbeing and

reduce the GBD (Global Burden of Disease) and hence my interest in this matter.

I should particularly like to mention here the burden to family members particularly if there

are cardiovascular & stroke complications. Apart from suffering individual the intangible

costs of pain, anxiety, inconvenience, lower quality of life and absenteeism from productivity

etc. Such loss cannot be quantified easily.

Finally above all the current days are of financial constraints and austerity. It is estimated

that the direct costs of the disease to UK's National Health Service are approximately 9% and

14% in USA to their total cost of healthcare budgets and is increasing (Type 2 Diabetes in

Practice; 2nd

Edition by Andrew J Krentz & Clifford J Bailey; Royal Society of Medicine

Press). The USA Healthcare Budget is $2 Trillion (More Health for the Money; Chapter 4 of

the World Health Report 2010

69

http://www.who.int/health_financing/strategy/equity_efficiency/en/). The Diabetes cost £1.5

million/ hour to NHS UK or 10% NHS Budget England and Wales (Diabetes UK) or

£25000/minute and total £14 billion/year for treating diabetes and its complications. The

WHO has in its fact sheet No 236 brought these costs out in terms of direct costs in

management of condition in direct costs & indirect costs e.g. effects on families, increased

insurance costs. Costs of lost production etc. wonderfully which makes a good reading (e.g.

in UK cost from Absenteeism being £8.4 Billion, early retirement £6.9billion/year & social

benefits £0.152billion - simply unimaginable (Costs of Diabetes

http://www.diabetes.co.uk/cost-of-diabetes.html).

The current increase is nearly 40%. This is a painful luxury no country can afford, especially

countries like India, China, the relatively low income countries. The condition can cost a

family 25% of its income for adult + diabetes & in USA 10% if there is a child diabetic and

worse so in African countries(Costs of Diabetes http://www.diabetes.co.uk/cost-of-

diabetes.html; Diabetes: the costs of diabetes Fact sheet N°236;

http://www.who.int/mediacentre/factsheets/fs236/en/). As one can clearly see, my reasoning

and interest in mitigating this chronic condition of Diabetes and its effects of

macrovasculature and microvascular affection now being the leading causes of Blindness due

to retinopathy and that of renal failure by the effects of nephropathy. Let alone the

neuropathy.

It is needless to impress upon the importance of regular monitoring of Blood Glucose if we

are to provide healthy life for diabetics and to avoid avoidable complications and their

consequences. As mentioned before attempts have been made to measure Blood Glucose by

traditional pinprick method to measure one off reading/normal procedure in diabetic current

management. Quick reading helps a diabetic to adjust his medications especially insulin

dosage regularly. After all it's a lifelong disease, the doctors and diabetic nurses are still not

available at our beck and call. What is more diabetes does not go on holidays even if patient

does. Hence diabetic needs to be educated himself about his condition and be strict

disciplinarian which is what Dr Bernstein was (the first patient to use glucometer) and

managed to control his sugars and avoid hypo and hyperglycaemia. Attempts to continuous

measurement of Blood Glucose as well as estimation by using non - invasive techniques,

though ideal has not been very successful and till these on off measures have to be mainstay

and of necessity has to be at the point of care namely near the patient (POCT method) and

best done by patient himself rather than to send blood to laboratory and wait for another 3 - 4

hours to get the result to adjust management of medications. This has not only decentralised

pathology work giving way to more personalised medicine. This is well reflected by the use

of portable small glucose monitors handled by individuals since its inception in late 60s -

early 70s of Ames Reflectance Meter (ARM).

Today the current market in US itself is more than 1 Billion and more than 3 billion

worldwide for the meters themselves let alone the no of billions of chips required world

wide/year which is a commended commercial output. Furthermore as mentioned previously

most of the techniques is utilising only 0.1% of the share with vast scope for further

70

expansion. Hence this is out of the reasons to work for such glucometers and make efforts

for continuous improvement which brings me to the final part of my rationalisation process

namely the proposed technique. However one has to remember the constant improvement

upon the existing conditions is the essence of mankind and thus a little dig into the history of

glucose measurement would be pertinent at this point.

2.2 History of Glucometer:

Clark's first experimental O2 electrode was produced in 1950. More than decade later in

1962 Clark and Lyon introduced the method for analyte measurement measuring the oxygen

consumption across the membrane protecting the electrode. As per the history record of

History Committee, Institute of Biomedical Science (IBMS) London; S.F.Clarke &

J.R.Foster 2012. Anton Clemens at Ames Research Division, Miles Laboratories, in Elkhart,

Indiana, USA (Later incorporated into Bayer Corporation) was the inventor of the first

glucometer prototype in 1968 and patient was issued on September 14th

1977 for its

manufacture. This was the 1st Ames Reflectance Meter (ARM) and the first patient to use it

was Dr Richard Bernstein (Dick Bernstein) who was type 1 Diabetic with fluctuant blood

sugar levels. The meter weighed 3lbs (due to heavy acid batteries) and cost $650 which was

a month's salary then. (Ref: Mendoza, D. (1999), Blood Glucose Meters, available at:

www.mendosa.com/bgmeters.htm (accessed 07/2014). The meter used Dextrostix to see

colour change to get from the chart approximately glucose levels. Today we have come a

long way. Since advent of Amperometry and Enzymes physiology; quite accurate readings

of glucose levels are achieved. The glucometer size is gone down considerably and it has

become a pocket sized instrument to carry. There still remain some problems as said above

of Interference & fouling which need to be overcome and hence my proposed methods to

correct this and get better meters by the proposed method above. Above also they need to be

cost effective as well.

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Figure 17: Anton H. Clemens (right) was the inventor of the first blood glucose meter while he

was the director of the Ames Instrument R&D Department of the Ames Division of Miles

Laboratories Inc. in Elkhart, Indiana. Here he is shown talking to George W. Orr, Jr., Group

Vice President, Professional Products Group (left) and Walter Ames Compton, M.D., President

and Chief Executive Officer of Miles Laboratories Inc. (center). This photograph appeared in

the 1969 Annual Report of Miles Laboratories Inc., Mendosa, D. (11/1999), Blood Glucose

Meters, available at: www.mendosa.com/

bgmeters.htm (accessed 07/2014).

My attempt using a carbon composite has 2 reasons:

1. First the individual component retain their properties e.g. metallic carbon graphite

would retain its electrical & mechanical, conductive properties and would produce

high signal to noise ratio to overcome interference would have low electrical

resistivity, pure crystal structure with low residual current. What is more making use

of composite will give it a distinct chemical, mechanical and physical properties that

are useful.

2. Secondly all these materials including membrane materials such as one in my propose

method namely Nafion have already been tested at Cranfield University in recent past

(Ref: J. D. Newman; Anal.Chem.1995).

Hence Nafion could give best possible precaution against interference along with PEG

(Polyethylene Glycol) coating to prevent fouling. MCA4 powder use would also offset costs

by having to use lower voltage of approximately 300V. Hence my proposed method for

glucometer. This makes sense to extend the same work experience as extended continuation

of project.

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CHAPTER 4: DESIGN OF DEVICE

1.1 Apparatus – Electrodes Construction

A three electrode Amperometric system consisting of Reference electrode of Ag/AgCl

working electrode modified carbon as described below and carbon counter electrode.

Construction of working electrode

Screen printed electrodes were used in this investigation, screen printer (DEK 247, DEK

printing machines, Weymouth, UK)multistage screen printing process was applied.

A silver conducting track (Acheson Colloids UK) was placed on a PVC substrate

(Genotherm, Sericol, Surrey, UK) previously cleaned with ethanol. This is a good supporting

polymer for electrode. A graphite pad (Electric dag 423SS, RFU) Acheson colloids,

Plymouth, UK) was printed at one end of silver track. An insulation layer (Matt Vinyl White

MV 27 Apollo colours, London, UK) was deposited over these layers to leave contact pad at

one end and working electrode (approximately 8mmX 2mm) at other end exposed. The

printed layer in each case use cured for 1 hour at 400C.

A variety of metallized carbon powders are available. My initial idea was to use Rhodinized

carbon Powder (MCA4) initially tested at Cranfield University labs and found very suitable

(Royston, UK). Unfortunately as the production of this has stopped. I intend to substitute

Platinized carbon. The powder mixed with a 2.5% (w/v) hydroxyethylcellulose (HEC, Fluka,

Gillingham, UK). The solution was dissolved in 0.1M phosphate buffer. The powder was

incorporated in HEC solution in the proportion 1:2 (w/w). The prepared ink was mixed using

rotary stirrer to get homogenous consistency for 1 hour at room temperature of 250C. The ink

was screen printed onto graphite pad.

1.2 Reference Electrode:

Ag/AgCl electrode is used as reference electrode in many commercial (BGM) strips. This

can be done in my Reference electrode as well using screen printing with Ag/AgCl ink using

polyester as a binding agent – the electrode reactions are as summarized

Glucose → – Glucanolactone + 2H+ + 2e-

AgCl + e- →Ag + Cl-

Glucose + 2AgCl → - Glucanolactone + 2Ag + 2H+

(Vasistha, et.al, 2011)

1.3 Counter Electrode

Which is also called auxiliary electrode is used only to make connection to electrolyte, so as

to be able to apply current to working electrode. This usually is made of inert material, non-

dissolvable. Hence I have chosen Graphite/carbon for this.

Sometimes to get graphite film adherent to PVC support a special solvent system (1.5% m/m

solution of cellulose acetate 1+1 (v/v) mixture of cyclohexanone and acetone can be used)

73

1.4 Enzyme Electrode Construction:

The enzyme predetermined is as said above is glucose oxidase (in our case Glucox PS, ABM,

Stockport, UK). The enzyme was mixed with catalytic powders to form printable ink print

prior to screen printing of working electrode. The formulation used being 1g of 2.5 %( w/v)

HEC in phosphate buffer (pH 7.0), 100mg of Glucox + 400mg of catalytic graphite powder).

After printing all electrodes are left to dry for at least 2hours at room temperature

(approximately 20 - 250C) (Jeffrey N et.al, 1995)

Above procedures of platinization of carbon by mixing of the metal in the carbon paste prior

to printing procedures, makes highly catalytic surface. Addition of enzyme makes the

material highly specific for glucose but in complex samples such as blood which can contain

other reducing sugars, uric acid, acetaminophen (paracetamol) etc. which are the interfering

compounds as mentioned in the discussion above (Section). Hence the use of membrane

once again to remind us of interference problem apart from the fact the the organic binders

used are soluble.

1.5 Construction of Membrane layer:

My initial idea was to use Naffion but this is expensive and thus difficult to procure. In order

to reduce cost I decided to use another good and commonly used non conducting material,

cellulose acetate 2% solution in acetone mixed with tetrabutylammonium, Toluene Sulfonate

(TBATS). As mentioned above in construction of working electrode, acetone is one of the

components of a solvent system in case of problem of adherence of graphite to PVC support

polymer and would thus serve this purpose as well.

For construction, ink jet printing technique was used (Biodot printer, Biodot, Diddington,

UK). This forces the prepared membrane fluid under high pressure through small nozzle

(75µm). the piezoelectric crystal which oscillates at high frequency of approximately

64000Hz, by producing shock waves to brake the jet fluid passing through the printer into

fine regular droplets. A charged electrode controlled by microprocessor controls the printing

process.

Buffer system for Glucose Biosensor test procedures

There are 3 buffer systems which can be used

PH 5 -5.5 0.1M Sodium Acetate

PH 6 – 7.5 0.1M sodium phosphate

pH 8 – 8.9 0.1M Tris HCl

As I have mentioned before Glucose Oxidase can work under wide range of pH 2.7 – 8.5);

the preferred buffer is 0.1M phosphate buffer pH 7.0 containing 0.1M KCl as normal pH of

blood values between (7.35 – 7.45) and it is for checking the blood Glucose, mainly that I am

constructing the Biosensor.

74

1.6 Preparation of Buffers solution:

As Buffer zone required to maintain pH (in our case intended pH 7.0) preparation of buffer is

an important step

Constituents & Method:

0.1M KH2PO4 (Potassium Di hydrogen phosphate) 500ml

0.1M K2HPO4 (Di Potassium Hydrogen Phosphate) 500ml

Using pH Electrode and under stirred conditions at room temperature 250C. pH achieved

=6.8.

Add 7.98grams of Di Sodium Hydrogen orthophosphate anhydrous & 6.9g of sodium di

hydrogen phosphate monohydrate under stirred conditions at room temperature (250C).

Results:

1000ml (1litre) buffer solution pH achieved =6.8.

I should like to point out that the buffers usually in solution in pairs. As the acidity (PKa

values) of all 4 constituents remain close between (12-13) (NaOH pKa =13, K2 HPO4 PKa

=12.4 and basicity pkb =6.8 and KH2PO4pKa =12.35). I believe they make good matching

Buffer Pairs.

1.7 Preparation of Glucose Solutions for Testing

Aim:

To make glucose solutions. 100mM, 10mM, 5mM, 1mM

Constituents & method

1. 100 grams of (β-D) Glucose (Sigma Aldrich) + Distilled water = (100ml or) under

stirred condition at Room Temp = 250C for

2. 10mM = 10ml glucose solution + 90ml Buffer for

3. 5mM = 5ml glucose solution + 95 ml Buffer for

4. 1mM = 1mM = 1ml glucose solution + 99 ml Buffer

All solutions prepared under stirred conditions at room temperature of 250C.

1.8 Experimental Method and Application of Membranes

As mentioned above in this section (Design of Device) array of 10 screen printed electrodes

(sensors) was prepared of which 2 were cut separately and named sensor 1 & sensor 2.

Carbon Graphite Ink (c2030408 P3, Gwent Group) designed to be used for screen printing

working electrodes (this gives superior electrochemical performance with Meldola’s blue and

contains Cobalt Phthalocyanine as mediator making it suitable for use with oxidase

enzymes). This paste was added onto the centre of electrode and allowed to dry

approximately 2 Hours.

Next step – GOx (Glucose Oxidase enzyme) 100mg in 1ml of buffer solution which was

pipetted onto the electrodes and allowed to dry for approximately 1hour.

Next step – 10ml (2%w/v) Acetone + 10ml cyclohexanone (2%w/v) +0.4grams Cellulose

Acetate.

75

Next step – applied this membrane material (Cellulose Acetate) to sensor 1 which was thick

membrane (approximately 250µm) and sensor 2 by lessening the concentration. Thin

membrane (approximately 100 µm) layers and allowed to dry overnight in the fume cupboard

this thus leaves 100µl of cellulose acetate solution added as a membrane over the electrode.

Further drying done in fume cupboard for approximately 20 minutes and there and then by

putting it in the oven for 1 hour.

The sensors were connected to Auto lab Electrochemical Analyser (Ecochemie, Utrecht,

Netherlands). All experiments performed under stirred conditions at room temperature 250C.

Dynamic current response monitored an

d recorded. First the blanks reading using only Buffer solutions. Followed by reading for

1mM, 5Mm, 10mM, & 100mM solutions. Reading tabulated and graphs of current Vs

Concentration plotted.

1.9 Equipment used:

Auto lab Electrochemical Analyser Sensor 1: Inserted in Buffer Solution

Sensor 1: Attached to the Auto Lab Sensor 1& 2: In fume hood; added the paste

76

Diagram of Electrode pH electrode

Screen Printed Electrodes

Sensor 1& 2 in oven to dry

77

CHAPTER 5: RESULTS FROM DEVICE

1.1: Sensor 1 Buffer

1.2: Sensor 1 1mM

1.3: Sensor 1 5mM

78

1.4: Sensor 1 10mM

1.5: Sensor 1: 100mM

79

X axis = Time in seconds

Y axis = Current in A

1.6: Current Vs Concentration for Sensor 1

X axis = Conc in (mM)

Y axis = Curent in (A)

Conc(mM) Current(A)

0 1

1 1.19

5 1.33

10 1.81

100 11.5

80

1.7: Sensor 2 Buffer

1.8: Sensor 2: 1mM

1.9: Sensor 2: 5mM

81

1.10: Sensor 2: 10mM

1.11: Sensor 2: 100mM

82

X axis = Time in seconds

Y axis = Current in A

1.12:

Conc Current

0 364

1 397

5 534

10 656

100 645

Concentration Vs Current for Sensor 2

83

CHAPTER 6: DISCUSSION/CONCLUSIONS

It is time now to take a stock of the whole exercise to realise what could have been done

better and what I could achieve the best under circumstances of multiple limitations. As one

would realise my initial idea of using Rhodinised powder of metalised carbon. MCA 4 had to

be given up as the company had stopped production. Even the application of Nafion

membrane had to be given up for the want of this expensive powder. The subsequent use of

that cellulose acetate membrane was as a compromise. Incidentally past experimentation at

Cranfield University Laboratories itself had proved the superiority of Nafion membrane over

cellulose acetate membrane so my idea of testing this fact and producing a near ideal

Biosensor was over. A worse factor was also the prohibition by time limit for

experimentation with fermentation media to produce more interfering species such as cells to

test the importance of membranes regards overcoming interference and fouling problems for

electrodes and to check whether my membrane application had made any difference regards

production of high signal to noise ratio. The basic intention of exercise of learning as to how

to apply membranes and produce electrodes practically in the laboratory was of course

achieved. As there was no availability of complex samples such as blood, no chance of

practical testing against interfering substances was possible.

It is however interesting from all the results obtained and from subsequent plotting of graphs

at different molar concentrations of glucose that the graphs have always remained linear

indicating that the amount of Dynamic current produced has remained directly proportional to

the concentration of glucose. This is the basic principle on which the amperometric

Biosensors are based. It is also to be noted that enzymatic reactions are not only specific but

are also generally quick under optimal conditions of pH and temperature control which is

why reactions last only few seconds and therefore sensors give quick results. The current in

amperometry is limited by diffusion of analyte to sensor surface. If one puts a membrane on

the electrode, it slows the rate of diffusion and hence the current. This also means that it

reaches saturation at lower analyte (Glucose) concentrations. It is important to note from

graphs that in sensor 2, much higher current is produced that in sensor 1 hence interpretation

is that as sensor 1 has thicker membrane, the rate of diffusion is much slower and hence

smaller current is produced. With sensor 2 membrane is thin, better diffusion and hence

higher current. Thus future amperometric devices should have thinner membrane. Finally it

gives a satisfaction to mind that I have learnt from the experiments as to how to produce an

enzymatic Biosensor using amperometric method in the laboratory and to get successful

results upholding basic principle that the concentration of analyte is directly proportional to

the amount of dynamic current produced.

84

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Appendices

Glucose Meter Test Working Strip Principle

89

Glucose Meter Block Diagram

90

Firmware Flowchart

91

Current to Voltage output with control solution high

Current to voltage converter output with control solution

normal

92

Current to voltage converter output with control solution low

93

Glucose meter demo board (front view)

94

U.A. NADKARNI MSc THESIS - Copy

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