UNIVERSITI PUTRA MALAYSIA

FARIBA JAFARI

FS 2012 30

IONIC CONDUCTIVITY AND DIPOLAR EFFECTS OF GLUCOSE ENZYMATIC SOLUTION AND FRUIT JUICES DETERMINED USING

DEVELOPED DUAL-FREQUENCY MICROWAVE GLUCOSE BIOSENSOR

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IONIC CONDUCTIVITY AND DIPOLAR EFFECTS OF GLUCOSE

ENZYMATIC SOLUTION AND FRUIT JUICES DETERMINED USING

DEVELOPED DUAL-FREQUENCY MICROWAVE GLUCOSE BIOSENSOR

By

FARIBA JAFARI

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirements for the Degree of Doctor of Philosophy

February 2012

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To My lovely Family,

The most wonderful gift from God.

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in

fulfillment of the requirement for the Degree of Doctor of Philosophy

IONIC CONDUCTIVITY AND DIPOLAR EFFECTS OF GLUCOSE

ENZYMATIC SOLUTION AND FRUIT JUICES DETERMINED USING

DEVELOPED DUAL-FREQUENCY MICROWAVE GLUCOSE BIOSENSOR

By

FARIBA JAFARI

February 2012

Chairman: Professor Kaida Khalid, PhD

Faculty: Science

A dual frequency microwave glucose biosensor is developed based on microwave

attenuation and is used for measuring the concentration of glucose content of fruit

juices. In this biosensor system two source frequencies 1GHz and 16.5GHz along

with the electronic switch were used to send the signal through the power divider to

the couple of microstrip sensors. Two wideband detectors were used to measure the

amplitude of the reflected signal from the sensor. The whole system was interfaced

to the labtop PC through the National Instruments data acquisition card, with the

help of software written in Labview graphical panel. It guides the user to perform

measurements easier and faster.

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This biosensor is suitable for a broad range of glucose content ranging from 0.01M

to 3M (Molar) which can help to control the quality of the juice in food industry. It is

based on the variation of the dielectric properties of the glucose solution after

reacting with the enzyme (GOx). Therefore the glucose content of solution sample

can be finding by measuring the attenuation of the signal through the sample.

The microstrip sensor was fabricated using RT-Duriod with dielectric properties of

2.2-j0.002 as the substrate, with operating frequency at 16.5 GHz. Using this type of

coaxial sensor only the small part of sample is needed to contact with the microstrip

line, therefore the measurement can be done with lower cost, more accuracy and in a

shorter time.

Theoretical analysis based on quasi-transverse electromagnetic mode (TEM mode)

in four layered microstrip is carried out to evaluate design parameters such as

microstrip characteristic impedance, effective dielectric constant, length and

thickness due to the sensitivity of the sensor. The analysis of the complex

electromagnetic waves in this system is presented using signal flow graphs and

solved by Mason's non-touching loops rules. To this end Visual Fortran programs is

written and documented to evaluate all the design parameters needed and to estimate

the microstrip patterns.

In the other part of this study, investigation was made to find the optimum ratio of

enzyme reaction with glucose solution and highest sensitivity of this technique, with

the studying of the dielectric properties of the glucose samples. The ionic

conductivity and dipolar effect on the polarization of the glucose solution after

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reacting with enzyme has been studied using two frequencies; one was below 2GHz

and one above that amount at room temperature.

In the purpose of verifying this sensing technique the results of the glucose solution

samples has been compared with the results for some type of fresh fruit juice

samples. It was found that the dielectric loss of the samples is affected by frequency,

concentration of glucose and ratio of enzyme reaction. The results of the different for

dielectric loss of the solution (glucose+ enzyme) and glucose, shows the changes are

preferable for derivation of glucose concentration.

The dual frequency biosensor was used to measure the attenuation and analysis of

the results at 1GHz and 16.5 GHz has been done completely. This biosensor tested

on four types of fruit juices in the range of 0.5M to 3.1M glucose concentration. The

biosensor has predicted glucose concentration with the accuracy of concentration

detection ±0.14 M using average method and can goes even up to ±0.13 M using

weighted average method. For the stability of this glucose biosensor, studying the

effect of operating time on the stability of this biosensor and enzyme behavior within

45 minutes of operating glucose at room temperature (25°C).

This microwave glucose biosensor has the limit of detection of glucose concentration

in solution in quiet higher than vivo measurement or medical application; therefore it

can be used more in food industry. Therefore the study of the development of the

dual frequency system using microwave technique will give benefit to the future

application of this technique application in food industries especially for products

such as juices, milk and etc, where both ionic conductivity and dipole effects are

considering simultaneously.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia

sebagai memenuhi keperluan untuk Ijazah Doctor Falsafah.

KESAN DIPOLAR DAN KEKONDUKSIAN IONIC LARUTAN GULKOSA

ENZYIM DAN JUS BUAH-BUAHAN YANG DITEKTUKAN DENGAN

MANGGUNAKAN BIOSENSOR GELOMBANG MIKRO KEKERAPAN

DUA

Oleh

FARIBA JAFARI

Februari 2012

Pengerusi: Profesor Kaida Khalid, PhD

Fakulti: Sains

Satu biosensor glukosa dua frekuensi gelombang mikro dibangunkan berdasarkan

pengecilan gelombang mikro dan digunakan untuk mengukur kepekatan kandungan

glukosa jus buah-buahan. Dalam sistem biosensor ini dua frekuensi sumber 1 GHz

dan 16.5 GHz bersama-sama dengan suis elektronik digunakan untuk menghantar

isyarat melalui pembahagi kuasa kepada beberapa sensor microstrip. Dua pengesan

Wideband digunakan untuk mengukur amplitud isyarat yang terpantul dari sensor.

Keseluruhan sistem di antara muka kepada komputer riba melalui pemerolehan kad

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data National Instruments, dengan bantuan perisian yang ditulis dalam panel grafik

Labview. Ia memberi panduan kepada pengguna untuk melakukan pengukuran lebih

mudah dan cepat.

Biosensor ini sesuai untuk julat kandungan glukosa antara 0.01M ke 3M (Molar)

yang boleh membantu untuk mengawal kualiti jus dalam industri makanan. Ia adalah

berdasarkan perubahan sifat-sifat dielektrik larutan glukosa selepas bertindak balas

dengan enzim (GOx). Oleh itu, kandungan glukosa dalam larutan sampel

penyelesaian boleh dicari dengan mengukur pengecilan isyarat melalui sampel.

Sensor mikrostrip telah direka dengan menggunakan RT-Duriod dengan sifat-sifat

dielektrik 2.2 j0.002 sebagai substrat, dengan frekuensi operasi pada 16.5 GHz.

Dengan menggunakan jenis sensor sepaksi ini hanya sebahagian kecil daripada

sampel diperlukan untuk menyentuh talian mikrostrip, Oleh itu, pengukuran boleh

dilakukan dengan kos yang lebih rendah, ketepatan yang lebih dan dalam masa yang

singkat.

Analisis teori yang berdasarkan kuasi-melintang elektromagnet (TEM mod) dalam

empat lapisan mikrostrip gunakan untuk menilai reka bentuk parameter seperti

impedans ciri mikrostrip, pemalar dielektrik berkesan, panjang dan tebal disebakkan

sensitiviti sensor. Analisis gelombang elektromagnet yang kompleks dalam sistem

ini dibentangkan menggunakan graf aliran isyarat dan diselesaikan dengan petua

gelung Mason tak menyentuh. Untuk tujuan ini program Visual Fortran program-

program ditulis dan didokumenkan untuk menilai semua parameter reka bentuk yang

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diperlukan dan anggaran corak mikrostrip.

Di bahagian lain kajian ini, penyiasatan telah dibuat untuk mencari nisbah optimum

tindak balas enzim dengan larutan glukosa dan sensitiviti tertinggi teknik ini, dengan

menghaji sifat-sifat dielektrik sampel glukosa. Kekonduksian ionik dan kesan

dwikutub pada polarisasi larutan glukosa selepas bertindak balas dengan enzim telah

dikaji dengan menggunakan dua frekuensi; salah satu berada di bawah 2 GHz dan

satu lagi di atas amaun itu pada suhu bilik.

Untuk mengesahkan teknik penderiaan keputusan ini sampel larutan glukosa telah

dibandingkan dengan keputusan untuk beberapa jenis sampel jus buah-buahan segar.

Ini didapati bahawa kehilangan dielektrik sampel dipengaruhi oleh frekuensi,

kepekatan glukosa dan nisbah tindak balas enzim. Keputusan yang berlainan bagi

kehilangan dielektrik larutan (glukosa + enzim) dan glukosa, menunjukkan

perubahan yang lebih baik untuk terbitan kepekatan glukosa.

Biosensor frekuensi dual telah digunakan untuk mengukur pengecilan dan analisis

keputusan di 1 GHz dan 16.5 G Hz telah dilakukan sepenuhnya. Biosensor ini diuji

ke atas empat jenis jus buah-buahan dalam lingkungan 0.5 M untuk 3.1 M kepekatan

glukosa. Biosensor meramalkan kepekatan glukosa dengan ketepatan pengesanan

kepekatan ± 0.14 M menggunakan kaedah purata dan boleh sehingga ± 0.13 M

menggunakan kaedah purata wajaran. Kestabilan glukosa juga dengan mengkaji

kesan masa operasi pada kestabilan biosensor dan tingkah enzim dalam masa 45

minit bagi glukosa yang beroperasi pada suhu bilik (25 ° C).

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Biosensor glukosa gelombang mikro ini mempunyai had pengesanan kepekatan

glukosa dalam tenang yang lebih tinggi daripada ukuran vivo atau aplikasi

perubatan, maka ia boleh digunakan dalam industri makanan. Oleh itu, kajian

pembangunan sistem dua frekuensi yang menggunakan teknik gelombang mikro

akan memberi manfaat kepada aplikasi masa hadapan teknik ini dalam industri

makanan terutamanya untuk produk seperti jus, susu dan lain-lain, di mana kedua-

dua kekonduksian ionik dan kesan dwikutub ditimbangkan serentah.

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ACKNOWLEDGEMENTS

"All praise to Almighty Allah, for his bounties and providences."

I would like to express my sincere gratitude to my supervisor, Professor Dr. Kaida

bin Khalid for his parentally guidance and advice during this research. His

encouragement, moral and technical support made this work possible.

I am also grateful to my supervisory committee, Associate Professor Dr. Jumiah

Hassan, Associate Professor Dr. Zulkifly Abbas and for their advice and helpful

discussion during this period of study.

I would like to thank:

Ø Mr. Mohd. Roslim and Mr. Hairi who have helped in

fabricating the patch and provided technical support in the

Laboratory.

Ø All the staff in physics department, UPM for their co-

operation given to me throughout my work.

Ø All of my lovely friends in Malaysia for enjoyable social life

in a wonderful country.

Last but not least, I wish to express my gratitude to my kind friends and my lovely

family for the support they gave throughout my studies. Long absent years from

home are often found with a warm and hopeful words.

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I certify that an Examination Committee has met on 13th

February 2012 to conduct

the final examination of Fariba Jafari on her thesis entitled “Ionic Conductivity and

Dipolar Effects of Glucose Enzymatic Solution and Fruit Juices Determinted Using

Developed Dual Frequency Microwave Biosensor” in accordance with the

Universities and University collegue Act 1971and the consititution of the Universiti

Putra Malkaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the

student be awarded the Doctor of Philosopy.

Members of the Examination Committee were as follows:

Abuld Halim bin Shaari, PhD

Professor

Faculty of Scienece

Universiti Putra Malaysia

(Chairman)

Wan Mohammed Daud bin Wan Yousoff, PhD

Associate Professor

Faculty of Scienece

Universiti Putra Malaysia

(Internal Examiner)

Alyani bint Ismail, PhD Faculty of Engineering

Universiti Putra Malaysia

(Internal Examiner)

Subhas C Mukhopadhyay, PhD

Professor

Massey University

New Zealand

(External Examiner)

SEOW HENG FONG, PhD Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date: 28 June 2012

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Doctor of Philosophy.

The members of the Supervisory Committee were as follows:

Kaida Khalid, PhD

Professor

Faculty of Science

Universiti Putra Malaysia

(Chairman)

Jumiah Hassan, PhD

Associate Professor

Faculty of Science

Universiti Putra Malaysia

(Member)

Zulkifly Abbas, PhD

Associate Professor

Faculty of Science

Universiti Putra Malaysia

(Member)

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations,

which have been duly acknowledged. I also declare that it has not been previously,

and is not concurrently, submitted for any other degree at Universiti Putra Malaysia

or other institutions.

FARIBA JAFARI

Date: 13 February 2012

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TABLE OF CONTENTS

Page

DEDICATION ii

ABSTRACT iii

ABSTRAK vi

ACKNOWLEDGEMENTS vii

APPROVAL ix

DECLARATION x

LIST OF TABLES xvi

LIST OF FIGURES xvii

LIST OF ABBREVIATIONS xx

CHAPTER

1 GENERAL INTRODUCTION

1.1 Introduction 1

1.2 Microwave Energy 4

1.3 History of Glucose Biosensor 6

1.4 Motivation, Objectives and Scopes of Research 7

1.5 Thesis Outline 8

2 THEORETICAL ANALYSIS BASED ON

MICROSTRIP STRUCTURE

2.1 Glucose Biosensor 11

2.2 Types of Glucose Biosensor 13

2.2.1 Thermal Method 13

2.2.2 Optical Method 15

2.2.3 Electrochemical Method 16

2.2.4 Piezo-electric (Resonant) Method 21

2.2.5 FET-based Method 23

2.3 Microwave Method for Glucose Detection 25

2.4 Enzyme Based Glucose Detection 26

2.4.1 Enzymatic Reaction of Glucose 28

2.4.2 Gluconic Acid 29

2.4.3 Hydrogen Peroxide 30

2.5 Limitation of Glucose Biosensing 32

2.6 Summary 33

3 THEORETICAL ANALYSIS BASED ON

MICROSTRIP STRUCTURE

3.1 Introduction 34

3.2 Structure of a Microstrip sensor 35

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3.3 Semi-infinite Double Covered Microstrip as Sensing Area 36

3.4 Stripline 39

3.5 The Total Attenuation of the Microstrip Sensor 40

3.6 Multiple reflection and Transmission at Multiple Interfaces 41

3.7 Calculation of Attenuation in the Sensing Structure

Based on System Signal Flow Graph 42

3.8 Results and Discussion 46

3.9 Properties of Materials 47

3.9.1 Polarization in Dielectrics Materials 48

3.9.2 Permittivity 51

3.10 Summery 53

4 SENSOR DEVELOPEMENT 4.1 Mictorstrip Glucose Biosensor Dual Frequency Set-up 56

4.2 Specification of Microstrip Biosensor 59

4.3 Calculation of Attenuation of the Whole Biosensor System 61

4.4 Summary 64

5 METHODOLOGY

5.1 Introduction 65

5.2 General Description of the Dual Frequency System 66

5.3 Voltage Controlled Oscillator, VCO 68

5.4 Low Barrier Schottky Diode Detector 69

5.5 Dual Coaxial Directional Coupler 70

5.6 The Electronic Solid State Switches 72

5.7 Power Divider 73

5.8 Interfacing and Graphical User Interface Program 73

5.9 Determination of Dielectric Properties 74

5.9.1 Calibration Procedures 76

5.9.2 Methodology 77

5.10 Standard Method of Glucose Content Measurement 78

5.11 Measurement of Glucose Content of the solutions

using ANA 79

5.11.1 Calibration Procedure for Attenuation Measurement

5.11.2 Experimental Set-up

5.12 Sample Preparation 81

5.13 Summery 84

6 RESULTS AND DISCUSSION

6.1 Dielectric Properties of Glucose 85

6.2 Dielectric Properties of Material of the Glucose Reaction

with Enzyme 88

6.3 Optimizing the Best Ratio of Glucose Reaction with Enzyme 89

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6.4 Dielectric Properties of the Mixture of Glucose-GOx Solution 91

6.5 Fruit Juices Samples 95

6.6 Calibration of Microstrip Glucose Biosensor 97

6.6.1 Effect of Operating Frequency 97

6.6.2 Effect of Ratio of Glucose Reaction with GOx 102

6.7 Glucose Detection Using Dual Frequency Set-Up 103

6.8 Glucose Detection of Fruit Juice Using Developed

Microstrip Biosensor 107

6.9 Stability of the Biosensor 113

6.10 Calibration and Analysis of Results for Dual

Frequency System 115

6.10.1 Calibration and Analysis at Single Frequency 115

6.10.2 Calibration of Biosensor Using the Average

Method of Prediction 117

6.10.3 Calibration of Biosensor Using the Weighted

Average Method 119

6.11 Summary 122

7 CONCLUSION AND FUTURE WORK 7.1 Conclusion 124

7.2 Future work 128

7.2.1 Determination of Sucrose in Fruit Juices 128

7.2.2 Determination of Galactose and Lactose Milk 130

REFERENCES 131

APPENDICES 141

BIODATA OF THE STUDENT 190