ANALYTICAL APPLICATION OF FUNCTIONALIZED CdS AND ZnS AS
FLUORESCENCE LABEL FOR THE DETERMINATION OF PROTEINS
SHEMALAH A/P RAMASUNDRAM
UNIVERSITI TEKNOLOGI MALAYSIA
ANALYTICAL APPLICATION OF FUNCTIONALIZED CdS AND ZnS AS
FLUORESCENCE LABEL FOR THE DETERMINATION OF PROTEINS
SHEMALAH A/P RAMASUNDRAM
A thesis submitted in fulfilment of the
requirements for the award of the degree of
Master of Science (Chemistry)
Faculty of Science
Universiti Teknologi Malaysia
DECEMBER 2005
iii
Specially dedicated to my beloved Ganesha appa, mum, dad, brother, sister and nada.
iv
ACKNOWLEDGEMENT
First and foremost, it’s my great pleasure to express my sincere appreciation
to my research supervisor, Assoc. Prof. Dr. Mustaffa Nawawi, who assisted and
guided me through this entire study. I would like to express my deepest gratitude for
his excellent scientific guidance, infinite patience and cooperation throughout this
work.
I am also very grateful to all academic staffs and the laboratory assistant of
the Department of Chemistry, Universiti Teknologi Malaysia especially En. Mat
Yasin for the technical assistance. I am grateful to the postgraduate students and all
my dear friends for their help and kindness. I am indebted to PTP- Universiti
Teknologi Malaysia for their financial support which made this academic study
possible.
The continuous encouragement and moral support from my beloved family
members is highly appreciated. Finally yet importantly, I would like to thank God for
giving me the will power and strength to accomplish this project. May this work
glorify His Name.
v
ABSTRACT
The quantitative analysis of protein is essential in biochemistry and clinical
medicine. The most sensitive quantitation of protein at this present is generally based
on fluorescence enhancement on organic dyes determination. However, this organic
fluorophores often suffer from photobleaching and low signal intensity. In order to
overcome such problems, the study was carried out to investigate the possibility of
employing the luminescent particle functionalized CdS and ZnS for quantitative
analysis of protein. CdS have been prepared and capped with mercaptoacetic acid
(functionalized CdS) whereas ZnS was capped with cysteine (functionalized ZnS),
which renders the particles water soluble and biocompatible. Fluorescence studies
showed at excitation wavelength Ȝexc = 233 nm, the maximum emission wavelength
of functionalized CdS was at 350 nm whereas for functionalized ZnS was at 357 nm
wavelength. Further, general optimization procedure such as the effect of pH,
temperature, concentration, reaction time of the functionalized CdS and
functionalized ZnS binding with BSA (Bovine Serum Albumin) was conducted. A
positive correlation with R2 = 0.9899 was obtained between CdS capped with
mercaptoacetic acid binding with BSA meanwhile correlation between ZnS capped
cysteine and BSA was 0.9805. The interferences of various metal ions and surfactant
were subsequently performed in order to obtain the selectivity of the developed assay
on the determination of BSA. The effect of surfactant such as ionic detergent sodium
dodecyl sulfate (SDS), nonionic detergent Triton X-100 shows signification shift
towards a shorter wavelength. Limit of detection for functionalized CdS binding with
BSA was 0.14 ppm followed by limit of detection of functionalized ZnS binding
with BSA was 0.09 ppm. This developed method was successfully applied to the
several types of protein such as egg albumin, lysozyme and amylase. The developed
novel assay is simple, inexpensive, rapid and sensitive.
vi
ABSTRAK
Analisis kuantitatif bagi protein adalah salah satu bidang yang sangat penting
dalam biokimia dan perubatan. Pada masa kini kaedah analisis kuantitatif bagi
protein lazimnya berdasarkan kaedah penentuaan pengikatan secara pendarflor
dengan bahan pewarna organik. Walau bagaimanapun kaedah ini didapati
mempunyai kelemahan dari segi penyahlunturan warna bahan organik pewarna and
kadar keamatan pendarflor yang rendah. Bagi mengatasi masalah ini, kajian
mengenai penggunaan partikel berpendarflor seperti CdS dan ZnS telah dijalankan
bagi menentukan kandungan protein. Dalam kajian ini, CdS telah diubahsuai dengan
menggunakan asid mercaptoasetik manakala ZnS telah diubahsuai dengan asid
amino sisteina bagi membolehkan partikel CdS dan ZnS bersifat larut dalam air dan
mudah bertindakbalas dengan komponen biologi. Kajian pendarfluor menunjukkan
pada puncak penyerapan Ȝex= 233 nm, puncak pemancaran telah diperolehi pada
Ȝem=350 nm bagi CdS dan Ȝem=357 nm bagi ZnS. Kajian selanjutnya seperti
pengoptimuman kesan pH, suhu, kepekatan dan masa tindakbalas bagi pengikatan
protein dengan CdS dan ZnS telah dilakukan. Graf kalibrasi linear diperolehi bagi
penentuan protein dengan CdS adalah R2=0.9899 dan dengan ZnS adalah R2=0.9805.
Kesan gangguan seperti bahan bukan protein, ion serta surfaktan telah dilakukan bagi
menentukan
kejituan
penentuan
kuantitatif
BSA.
Kajian
kesan
surfaktan
menunjukkan surfaktan ionik iaitu natrium dodesil sulfat (SDS) dan surfaktan bukan
ionik iaitu Triton X-100 menunjukkan puncak pemancaran beranjak ke panjang
gelombang yang lebih rendah. Had pengesanan bagi penentuan protein dengan CdS
adalah 0.14 ppm manakala bagi ZnS adalah 0.09 ppm. Kaedah yang dibangunkan
telah diaplikasikan bagi penentuaan pelbagai protein seperti albumin telur, lisozim
dan amilase. Daripada hasil kajian yang telah dilakukan didapati bahawa kaedah
penentuan protein yang dibangunkan adalah mudah, murah, pantas and sensitif
berbanding dengan kaedah lain.
vii
TABLE OF CONTENTS
CHAPTER
1
TITLE
PAGE
TITLE PAGE
i
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGEMENT
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
xiii
LIST OF FIGURES
xiv
LIST OF SYMBOLS
xix
INTRODUCTION
1
1.1
Prelude
1
1.2
General Introduction to Proteins
2
1.2.1
Classification of Proteins
3
1.2.1.1 Simple Proteins
3
1.2.1.2 Conjugated Proteins
3
1.2.1.3 Derived Proteins
4
Proteins Structure
4
1.2.2
1.3
Bovine Serum Albumin
5
1.4
Functions of Proteins
6
1.4.1
Enzymatic Catalysis
6
1.4.2
Transport and Storage
6
1.4.3 Immune Protection
1.4.4
Generation and Transmission of Nerve
6
viii
1.5
Impulses
7
1.4.5
Control of Growth and Differentiation
7
1.4.6
Contribution to Acid Base Balance
7
1.4.7
Providing Energy
8
The Basic Protein Quantitation Protocols
8
1.5.1
Colorimetric Method
8
1.5.1.1 Biuret Method
9
1.5.1.2 Lowry Method
9
1.5.1.3 Bradford Method
10
1.5.2 Bicinhoninic Acid (BCA) Method
10
1.5.3 Kjeldahl Method
11
1.5.4 UV Absorbance Method
12
1.6
Recent Advances in Protein Analysis
12
1.7
Fluorescence Method in Proteins Analysis
15
1.7.1
Luminescent Organic Compounds
Labeling Proteins
1.7.2
16
Luminescent Semiconductor Labeling
Biomolecules
19
1.7.2.1 Cadmium Sulfide Labeling
Biomolecules
20
1.7.2.2 Zinc Sulfide Labeling
Biomolecules
1.8
2
22
Advantages and Disadvantages of Fluorescence
in Protein Analysis
23
1.8.1
Disadvantages
24
1.9
Research Background
25
1.10
Research Objective and Scope of Study
26
EXPERIMENTAL
27
2.1
Apparatus
27
2.2
Reagents
28
2.3
Preparation of Standard Solutions
29
2.3.1
Preparation of 0.1 mol L-1 Cadmium
Chloride (CdCl2.H2O)
29
ix
2.3.2
Preparation of 0.1 mol L-1 Sodium Sulphide
(Na2S.9H2O)
2.3.3
Preparation of 1.0 mol L-1 Zinc Sulphate
(ZnSO4)
2.3.4
Preparation of Stock Solutions
2.4.1
30
Preparation of Mercaptoacetic acid
1.0 mol L-1
2.4
29
Preparation of Hydrochloric Acid (HCl)
0.01 mol L-1
2.3.6
29
Preparation of 1.0 mol L-1 Tris buffer
(tris-hydroxymethyl aminomethane)
2.3.5
29
30
30
Preparation of Bovine Serum Albumin
(BSA), Albumin, Lysozyme, Amylase
Stock Solution (100 ppm)
2.5
30
Procedure for Preparing CdS Capped
Mercaptoacetic Acids
30
2.6
Procedure for Preparing ZnS capped Cysteine
31
2.7
Fluorimetric Analysis
31
2.8
UV-Vis Spectroscopy Analysis
32
2.9
General Procedures
32
2.9.1
32
Effect of pH
2.9.2 Effect of Buffer
33
2.9.3
33
Effect of Concentration
2.9.4 Effect of Temperature
33
2.9.5
Effect of Reaction Time
34
2.9.6
Calibration Curves for the Quantitative
Analysis of BSA
34
2.9.7 Accuracy of the Method
34
2.9.8
Limit of the Detection and Quantification
35
2.9.9
Salt–Dependent Studies on Functionalized
CdS Binding with BSA
35
2.9.10 Effect of Foreign Substances
35
2.9.11 Effect of Surfactant
36
x
3
FUNCTIONALIZED CADMIUM SULFIDE BINDING
WITH BSA
37
3.1
Preamble
37
3.2
Fundamental Studies of CdS Capped Mercaptoacetic
Acid
(Functionalized CdS) Particles
3.2.1
Formation of CdS Capped with
38
Mercaptoacetic Acid (Functionalized CdS)
Particles
3.2.2
38
Fluorescence Properties of CdS Capped with
Mercaptoacetic Acid (Functionalized CdS) 39
3.3
Fluorescence Properties of Functionalized CdS
Binding with BSA
3.3.1
40
Standard Calibration of Absorbance
Properties on (Functionalized CdS)
Binding with BSA
3.3.2
40
Fluorescence Properties of the CdS Capped
Mercaptoacetic Acid (Functionalized CdS)
Binding with BSA
3.4
42
Optimization Procedures on the Fluorescence
Properties of the Functionalized CdS Binding
3.5
with BSA
44
3.4.1
Effect of pH Value
44
3.4.2
Effect of Buffer Nature
45
3.4.3
Effect of Concentration
46
3.4.4
Effect of Temperature
47
3.4.5
Effect of Reaction Time and Stability
48
Standard Calibration Curves for the Quantitative
Analysis of Functionalized CdS Binding with BSA 49
3.6
Method Validation for the Quantitative Analysis of
Functionalized CdS Binding BSA
51
3.6.1
Linearity of Standard Curves
51
3.6.2
Accuracy of the Method
52
3.6.3
Limit of the Detection and Quantification
53
xi
3.7
Salt-Dependent Studies on Functionalized
CdS Binding with BSA
3.8
3.9
Effect of the Foreign Substances on
Functionalized CdS Binding with BSA
55
3.8.1
Amino Acids
56
3.8.2
Carbohydrates
57
3.8.3
Metal ions
58
3.8.4
Other compounds
59
3.8.5
Surfactant
61
Standard Calibration of Proteins Binding with
Functionalized CdS
3.10
3.11
4
54
64
Comparison of the Developed Functionalized CdS
Methods
68
Conclusions
69
FUNCTIONALIZED ZINC SULFIDE BINDING BSA 70
4.1
Preamble
4.2
Fundamental Studies of ZnS Capped L-Cysteine
(Functionalized ZnS)
4.2.1
72
Fluorescence Properties of ZnS Capped L -Cysteine
(Functionalized ZnS) binding with BSA
4.5
71
Standard Calibration of Absorbance Properties
on Functionalized ZnS Binding with BSA
4.4
71
Spectral Characteristics of Fluorescence on
Functionalized ZnS
4.3
70
74
Optimizations Procedures on the Fluorescence
Properties of the Functionalized ZnS Binding with
4.6
BSA
76
4.5.1 Effect of pH Value and Buffer Nature
76
4.5.2
Effect of Concentration
78
4.5.3
Effect of Temperature and Reaction Time
79
Standard Calibration Curves for the Quantitative
Analysis of Functionalized ZnS binding with BSA 81
4.7
Method Validation for the Quantitative Analysis
xii
of BSA
4.7.1
Linearity, Accuracy, Limit of the Detection
of the Method
4.8
4.10
4.11
5
83
Salt Dependence Studies on Functionalized ZnS
Binding with BSA
4.9
83
84
Effect of the Foreign Substances on Functionalized
ZnS Binding with BSA
86
4.9.1 Effect of Non-protein Substances
86
4.9.2
The Effect of Surfactant
90
4.9.2.1 Sodium Dodecyl Sulfate (SDS)
90
4.9.2.2 Triton X-100
91
Standard Calibration of Proteins Binding with
Functionalized ZnS
92
Conclusions
97
CONCLUSIONS AND SUGGESTIONS
98
5.1
Conclusions
98
5.2
Suggestions
101
REFERENCES
102
xiii
LIST OF TABLES
TABLE NO.
TITLE
PAGE
1.1
Amino acid composition of BSA
5
3.1
The effect of different type of buffer in the presence
and absences of BSA. pH 6, [BSA] = 25 ppm,
[Functionalized CdS colloids] = 3x 10-4 mol L-1
46
Intra-day accuracy of functionalized CdS binding with
BSA under optimum conditions. pH 6, [Functionalized
CdS colloids] = 3x10-4 mol L-1
52
Inter-day precision of functionalized CdS binding with
BSA under optimum conditions. pH 6, [Functionalized
CdS colloids] = 3x10-4 mol L-1
52
Effect of foreign substances on the fluorescence intensity
of functionalized CdS binding with BSA 25 ppm
60
Analytical parameter of various proteins binding
with functionalized CdS under optimum conditions.
pH 6, [Functionalized CdS colloids] = 3x10-4 mol L-1.
65
Comparison of methods for the determination of protein
(BSA)
69
The effect of different type of buffer in the presence and
absences of BSA. pH 6, [BSA] = 15 ppm,
[Functionalized ZnS colloids] = 4x 10-4 mol L-1
78
3.2
3.3
3.4
3.5
3.6
4.1
4.2
Effect of foreign substances on the fluorescence
intensity of functionalized ZnS binding with BSA 15 ppm 87
4.3
Analytical parameter of various proteins binding
with functionalized ZnS under optimum conditions. pH 7,
93
[Functionalized ZnS colloids] = 4x10-4 mol L-1
xiv
LIST OF FIGURES
FIGURE NO.
TITLE
PAGE
1.1
General structure of Į-amino acids
2
2.1
Luminescence spectrometer Perkin-Elmer Model LS-50B 28
3.1
Yellow CdS solution capped with mercaptoacetic
acid
38
3.2
Fluorescence (a) excitation spectrum and (b) emission
spectrum for CdS capped with mercaptoacetic acid. Ȝexc =
233 nm, [Functionalized CdS colloids] = 3 x10-4 mol L-1 40
3.3
UV Absorbance spectra of functionalized CdS binding with
BSA with increasing concentration 0 ppm to 35 ppm.
41
[Functionalized CdS colloids] = 3x10-4 mol L-1
3.4
Standard calibration for functionalized CdS in the presence
of various concentration of BSA. . [Functionalized CdS
42
colloids] = 3x10-4 mol L-1
3.5
The emission spectra of functionalized CdS in the absence
and presence of BSA under the room temperature.
[BSA] = 25 ppm, [Functionalized CdS colloids] =
43
3x10-4 mol L-1
3.6
Effect of pH for functionalized CdS in the absence and
presence of BSA at room temperature. [BSA]=25 ppm,
[Functionalized CdS colloids] = 3x10-4 mol L-1
44
Effect of functionalized CdS concentration on the
fluorescence intensity in the presence of BSA. pH 6,
[BSA] = 25 ppm, [Functionalized CdS colloids] =
3x10-4 mol L-1
47
Effect of temperature for functionalized CdS in absence
and presence of BSA. pH 6 [BSA] = 25 ppm,
[Functionalized CdS colloids] = 3x10-4 mol L-1
48
3.7
3.8
xv
3.9
3.10
3.11
3.12
Effect of reaction time for functionalized CdS in the
presence of BSA. pH 6, [BSA] = 25 ppm,
[Functionalized CdS colloids] = 3x10-4 mol L-1
49
Emission spectra of functionalized CdS with increasing
concentration of BSA from 0 ppm to 50 ppm. pH 6,
[Functionalized CdS colloids] = 3x10-4 mol L-1
50
Calibration curves for functionalized CdS binding with
various concentration of BSA under optimum conditions.
pH 6, [Functionalized CdS colloids] = 3x10-4 mol L-1
51
Effect of the increasing concentration of BSA on the
functionalized CdS. pH 6, [Functionalized CdS colloids]
= 3x10-4 mol L-1
53
3.13
Effect of the concentration of NaCl solution on the
fluorescence intensity in the absence (curve 1) and presence
(curve 2) of BSA and functionalized CdS.BSA=25 ppm,
55
[Functionalized CdS colloids] = 3x10-4 mol L-1
3.14
Effect of increasing concentration of L-alanine, L-glycine
and L-cysteine on functionalized CdS binding with BSA.
pH 6, [BSA] = 25 ppm, [Functionalized CdS colloids]
= 3x10-4 mol L-1
56
Effect of increasing concentration of starch, sucrose and
glucose on functionalized CdS binding with BSA. pH 6,
[BSA] = 25 ppm, [Functionalized CdS colloids] =
3x10-4 mol L-1
57
3.15
3.16
Effect of increasing concentration of Ca2+, Cu2+ NH4+, Ni2+
on functionalized CdS binding with BSA. pH 6, [BSA] =
25 ppm, [Functionalized CdS colloids] = 3x10-4 mol L-1
58
3.17
Effect of increasing concentration of Fe2+, Fe3+ Zn2+, Mg2+
on functionalized CdS binding with BSA. pH 6, [BSA] =
59
25 ppm, [Functionalized CdS colloids] = 3x10-4 mol L-1
3.18
Effect of increasing concentration of EDTA and urea on
functionalized CdS binding with BSA. pH 6, [BSA] = 25
ppm, [Functionalized CdS colloids] = 3x10-4 mol L-1
60
Fluorescence emission spectra of CdS binding BSA
with increasing concentration of SDS 10 ppm-100 ppm.
pH 6, [BSA] = 25 ppm, [Functionalized CdS colloids] =
3x10-4 mol L-1
62
3.19
3.20
Effect of increasing of SDS concentration on the
fluorescence emission intensity on functionalized CdS
xvi
binding with BSA. pH 6, [BSA] = 25 ppm,
[Functionalized CdS colloids] = 3x10-4 mol L-1
63
3.21
Fluorescence emission spectra of functionalized CdS
binding with BSA with increasing concentration of
Triton X-100 from 10 ppm to 50 ppm. pH 6, [BSA]
= 25 ppm, [Functionalized CdS colloids] = 3x10-4 mol L-1 63
3.22
Emission spectra of functionalized CdS with increasing
concentration of lysozyme from 0 ppm to 12.5 ppm. pH 6,
[BSA] = 25 ppm, [Functionalized CdS colloids] = 3x10-4
mol L-1
65
3.23
Emission spectra of functionalized CdS with increasing
concentration of amylase from 0 ppm to 10 ppm. pH 6,
[BSA] = 25 ppm, [Functionalized CdS colloids] = 3x10-4
mol L-1
66
Emission spectra of functionalized CdS with increasing
concentration of egg albumin from 0 ppm to 30 ppm.
pH 6, [BSA] = 25 ppm, [Functionalized CdS colloids] =
3x10-4 mol L-1
66
Calibration graph for functionalized CdS binding with
protein lysozyme under the optimum conditions. pH 6,
[Functionalized CdS colloids] = 3x10-4 mol L-1
67
Calibration graph for functionalized CdS binding with
protein amylase under the optimum conditions. pH 6,
[Functionalized CdS colloids] = 3x10-4 mol L-1
67
Calibration graph for functionalized CdS binding with
protein egg albumin under optimum conditions. pH 6,
[Functionalized CdS colloids] = 3x10-4 mol L-1
68
Fluorescence excitation spectrum (a) and emission
spectrum (b) for ZnS capped cysteine. Ȝexc = 233 nm.
[Functionalized ZnS colloids] = 4x10-4 mol L-1
72
UV absorbance spectra of functionalized ZnS and BSA
concentration 0 ppm to 35 ppm. [Functionalized ZnS
colloids] = 4x10-4 mol L-1.
73
3.24
3.25
3.26
3.27
4.1
4.2
4.3
Standard calibration for functionalized ZnS in presence of
various concentration of BSA. [Functionalized ZnS colloids]
73
= 4x10-4 mol L-1.
4.4
The emission spectra of functionalized ZnS in the absence
and presence of BSA under the room temperature. [BSA]
= 15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1 74
xvii
4.5
4.6
Schematic of ZnS capped L-cysteine
Schematic of functionalized ZnS conjugated to protein
75
75
4.7
Effect of pH for functionalized ZnS in absence and
presence of BSA at room temperature.[BSA] = 15 ppm,
[Functionalized ZnS colloids] = 4x10-4 mol L-1
77
4.8
Effect of functionalized ZnS concentration on the
fluorescence intensity in the presence of BSA. pH 7, [BSA]
= 15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1 79
4.9
Effect of temperature for functionalized ZnS in absence
and presence of BSA . pH 7, [BSA] = 15 ppm,
[Functionalized ZnS colloids] = 4x10-4 mol L-1
80
Effect of reaction time for functionalized ZnS in the
presence of BSA. pH 7, [BSA] = 15 ppm,
[Functionalized ZnS colloids] = 4x10-4 mol L-1
81
4.10
4.11
Emission spectra of functionalized ZnS with increasing
concentration of BSA from 2.5 ppm to 20 ppm. pH 7, [BSA]
= 15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1 82
4.12
Calibration curves for functionalized ZnS binding with
various concentration of BSA under optimum conditions.
pH 7, [BSA] = 15 ppm, [Functionalized ZnS colloids] =
4x10-4 mol L-1
83
Effect of the increasing concentration of BSA on the
functionalized ZnS. pH 7, [BSA] = 15 ppm,
[Functionalized ZnS colloids] = 4x10-4 mol L-1
84
4.13
4.14
Effect of the concentration of NaCl solution on the
fluorescence intensity in the absence (curve 1) and presence
(curve 2) of BSA and functionalized colloidal ZnS. pH 7,
[BSA] = 15 ppm, [Functionalized ZnS colloids] =
85
4x10-4 mol L-1
4.15
Effect of increasing concentration of L-alanine, L-glycine
and L-cysteine on ZnS binding with BSA. pH 7, [BSA] =
15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1
88
Effect of increasing concentration of starch, sucrose and
glucose on functionalized ZnS binding with BSA. pH 7,
[BSA] = 15 ppm, [Functionalized ZnS colloids] =
4x10-4 mol L-1
88
4.16
4.17
Effect of increasing concentration of Mg2+, Cu2+ NH4+ and
Ni2+ on functionalized ZnS binding with BSA. pH 7,
xviii
4.18
4.19
4.20
[BSA] =15 ppm, [Functionalized ZnS colloids] =
89
4x10-4 mol L-1
2+
3+
2+
Effect of increasing concentration of Fe , Fe Cd and
Ca2+ on functionalized ZnS binding with BSA. pH 7, [BSA]
= 15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1 89
Effect of increasing concentration of EDTA and urea
on functionalized ZnS binding with BSA. pH 7, [BSA] =
15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1
90
Fluorescence emission spectra of ZnS binding with BSA
with increasing concentration of SDS 10 ppm-100 ppm.
pH 7, [BSA] = 15 ppm, [Functionalized ZnS colloids] =
4x10-4 mol L-1
91
4.21
Fluorescence emission spectra of functionalized ZnS
binding with BSA with increasing concentration of
Triton X-100 from 10 ppm to 30 ppm. pH 7, [BSA]
= 15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1 92
4.22
Emission spectra of functionalized ZnS with increasing
concentration of amylase from 0 ppm to 7.5 ppm. pH 7,
[Functionalized ZnS colloids] = 4x10-4 mol L-1
94
Emission spectra of functionalized ZnS with increasing
concentration of lysozyme from 0 ppm to 10 ppm. pH 7,
[Functionalized ZnS colloids] = 4x10-4 mol L-1
94
Emission spectra of functionalized ZnS with increasing
concentration of egg albumin from 0 ppm to 20 ppm.
pH 7, [Functionalized ZnS colloids] = 4x10-4 mol L-1
95
Calibration graph for functionalized ZnS binding with
protein amylase under optimum conditions. pH 7,
[Functionalized ZnS colloids] = 4x10-4 mol L-1
95
Calibration graph for functionalized ZnS binding with
protein lysozyme under optimum conditions.pH 7,
[Functionalized ZnS colloids] = 4x10-4 mol L-1
96
Calibration graph for functionalized ZnS binding with
protein egg albumin under optimum conditions. pH 7,
[Functionalized ZnS colloids] = 4x10-4 mol L-1
96
4.23
4.24
4.25
4.26
4.27
xix
LIST OF SYMBOLS
BRB
-
Britton-Robinson buffer
BSA
-
Bovine Serum Albumin
CdS
-
Cadmium Sulfide
HSA
-
Human Serum Albumin
EDTA
-
ethylenediaminetetraacetic
INT
-
Intensity
LOD
-
Limit of detection
nm
-
nanometer
ppm
-
parts per million
RSD
-
Relative standard deviation
SDS
-
sodium dodecyl sulfate
UV-Vis
-
Ultraviolet visible
ZnS
-
Zinc Sulfide
ı
-
Standard deviation
Ȝexc
-
Excitation wavelength
Ȝem
-
Emission wavelength
CHAPTER 1
INTRODUCTION
1.1
Prelude
Proteins have long been recognized as biologically fundamental and
medically important substance. International and industrial competition provided a
healthy impetus to basic research and produced an explosive growth in fundamental
understanding about protein molecules, which constituted the ‘stuff of life’ [1].
Proteins contributed to key body function, including blood clotting, fluid
balance, production of hormones and enzymes, vision and cell growth and repairs.
Day by day regulation and maintenance of the body requires thousands of different
proteins, which is a much greater variety than carbohydrates and lipids in the body.
Many of these proteins are very large and their protein molecular weights can exceed
one million. In contrast, glucose has a molecular weight of only 180. Thus, from a
biological standpoint, proteins deserved their name, from the Greek word proteios,
means, “To come first” [2].
2
1.2
General Introduction to Proteins
The name of protein (Greek, proteios) was first used in 1838 by Mulder
following a suggestion by Berzelius [3]. Mulder was among the first proposed a
systematic study of the elemental composition of proteins. Most proteins were found
to contain 50 to 55% carbon, 6 to 7% hydrogen, 20 to 23% oxygen and 12 to 19%
nitrogen. Protein determinations were based on nitrogen (assuming an average
content of 16%) came to be use for analysis of tissues and food samples. Sulfur (0.2 0.3%) was found to occur in proteins and phosphorus in some cases (as high as 3%).
Trace elements identified in certain proteins (e.g. 0.34% iron in hemoglobin)
permitted calculation of minimum molecular weights. These results give the first
indication that proteins have large molecular weight compared to other organic
substances known at that time [3].
During the late 1800s, amino acids were known as the basic building units of
proteins. There are 20 amino acids occurred as components of most proteins. In
1902, Fischer proposed that proteins are long chains of amino acids joined together
by amide bonds between the Į-carboxyl group of the one amino acid and the Į-amino
group of another [3]. The water was eliminated between the Į-carboxyl group of one
amino acid and the amino group of another to produce an amide linkage [3, 4].
Proteins also can be identified as natural polymer of L-Į-amino. Figure 1.1 shows the
general structure of the Į-amino acids.
Figure 1.1: General structure of Į-amino acids
3
1.2.1
Classification of Proteins
Proteins are classified in two additional ways as shape and composition.
Based on their shape, proteins can be classified into two major groups that are
fibrous protein and globular protein [1]. Fibrous proteins are insoluble and
characteristically play a structural role in animals. Typical member of fibrous group
are fibroin of silk, keratin of hair, nails and feather and elastin of elastic connective
tissue. The proteins, which resemble the white of an egg closely, such as proteins of
milk and blood plasma, are classified as globular proteins. This globular protein have
approximately spherical dimension, which is in contrast with fibrous proteins.
Globular proteins are characteristically soluble in aqueous solution [1].
On basis of composition, proteins are classified as simple proteins,
conjugated proteins and derived proteins [2].
1.2.1.1 Simple Proteins
Simple proteins yield only amino acids on hydrolysis. Albumin, globulin,
glutelins and protamines are classified as simple protein. Simple proteins usually are
proteins of relatively low molecular weight.
1.2.1.2 Conjugated Proteins
Conjugated proteins contain an amino acid part combined with a non-protein
material such as lipids, nucleic acids and carbohydrates. Some of the major
conjugated
proteins
chromoproteins.
are
phosphoproteins,
lipoproteins,
glycoproteins
and
4
1.2.1.3 Derived Proteins
Derived protein is protein that has been changed by physical agents such as
heat and high hydrogen-ion concentration and by action of enzymes or chemical
reagents. This protein can be divided into two groups by the amount of hydrolysis
that can take place. These groups are classified as primary and secondary derivatives.
1.2.2
Proteins Structure
Proteins are extraordinarily complex molecules. There are four levels of the
structural organization of proteins that have been distinguished, which are primary
structure, secondary structure, tertiary structure and quaternary structure [6, 3]. The
primary structure of proteins was related to the peptide bonds between the amino
acids component and to the amino acid sequence in the molecule. Proteins have been
defined as the linear sequence of amino acid residues making up the polypeptide
chains of the molecule [3].
A polypeptide chain may involve in hydrogen bonding between amide
nitrogen and carbonyl oxygen. These bonds may be formed between different areas
of the same polypeptide chain or between adjacent chains. Based on this bond,
secondary structure of proteins was established. The structure may be in two types
helical structure and sheet structure. The helical structures are stabilized by
intramolecular hydrogen bonds and the sheet structures by intermolecular hydrogen
bonds [6, 7].
The tertiary structure of proteins was established when the chains are folded
over into compact structures stabilized by hydrogen bonds, disulfide bridges and van
der Waals forces. Further, proteins contained more than one polypeptide chain
exhibit as an additional level of structural organization. Quaternary structure referred
to the spatial arrangement of subunit and the nature of their contacts [8].
5
1.3
Bovine Serum Albumin
Serum albumin is one of the most widely studied proteins and is the most
abundant protein in plasma with a typical concentration of 5 g/100 mL. Albumin
generally regarded to mean serum albumin or plasma albumin. Albumins are the
most abundant protein in the circulatory system and contribute 80 % to colloid
osmotic blood pressure [9]. Albumins have been used as a model protein for diverse
biophysical, biochemical and physicochemical studies.
Bovine serum albumin (BSA) is essentially a kind of plasma protein extracted
from bovine (buffalo blood). BSA built by a single chain of 528 amino acid globular
non-glycoprotein cross-linked with 17 cysteine residues (8 disulfide bonds and 1 free
thiols) [10]. Table 1 shows amino acid composition of BSA [12, 13]. Albumins
characterized by a low content of tryptophan and methionine and a high content of
cysteine and the charged amino acids, aspartic and glutamic acids, lysine and
arginine. The glycine and isoleucine content of BSA are lower than in the average
protein [11]. (Table 1)
It has now been determined that serum albumin is chiefly responsible for the
maintenance of blood pH [14]. Moreover, BSA has a wide range of physiological
functions involving the binding, transport and delivery of fatty acids, porphyrins,
tryptophan, tyrosine and steroids [10].
Table 1.1: Amino acid composition of BSA [12, 13]
Ala 48
Cys 35
Asp 41
Glu 58
Phe 30
Gly 17
His 16
Ile 15
Lys 60
Leu 65
Met 5
Asn 14
Pro 28
Gln 21
Arg 26
Ser 32
Thr 34
Val 38
Trp 3
Tyr 21
6
1.4
Functions of Proteins
Proteins play crucial roles in virtually all-biological process. Their
significance and the remarkable scopes of their activity are exemplified in the
following functions [1, 2, 8].
1.4.1
Enzymatic Catalysis
Most of the chemical reactions in biological systems are catalyzed by specific
macromolecules called enzymes. Enzymes exhibited enormous catalytic power and
usually reaction rates increased by at least a million fold. The striking fact is that
nearly all known enzymes are proteins.
1.4.2
Transport and Storage
Specific proteins transport most of the small molecules and ions. For
example, hemoglobin transports oxygen in erythrocytes, whereas myoglobin a
related protein, transports oxygen in muscle. Iron is carried in the plasma of blood
and transferred into the liver and stored as a complex with ferritin.
1.4.3
Immune Protection
Antibodies are highly specific proteins that recognize and combine with
foreign substances such as viruses, bacteria and cells from other organisms. Without
sufficient dietary protein, the immune system lacks the cells and other tools needed
to function properly.
7
1.4.4
Generation and Transmission of Nerve Impulses
Receptor proteins mediated the response of nerve cells to specific stimuli. For
example, rhodopsin is the light-sensitive protein in retinal rod cells. Receptor
proteins triggered by specific small molecules, such as acetylcholine is responsible
for transmitting nerve impulses at synapses (junctions between nerve cells).
1.4.5
Control of Growth and Differentiation
Control sequential expression of genetic information is essential for the
orderly growth and differentiation of cells. In bacteria, repressor proteins are
important control elements that silence specific segments of the DNA of the cell. In
higher organism, growth factor proteins control growth and differentiation. Besides
that, the activities of different cells in multicellular organisms are coordinated by
hormone. Many of the hormones, such as insulin and thyroid stimulating hormone
are proteins. Indeed, proteins served in all cells as sensors that control the flow of
energy and matter.
1.4.6
Contribution to Acid Base Balance
The concentration of hydrogen ions in the bloodstream determines the acid-
base balance (pH) of the blood. Proteins help to regulate the amount of free hydrogen
ions by readily accepting or donating hydrogen ions. This regulation helps to keep
the blood pH fairly constant and slightly alkaline (pH 7.35 to 7.45).
8
1.4.7
Providing Energy
Proteins supplies about 2% to 5% of the energy the body uses. Most cells use
primarily carbohydrates and fats for energy. Proteins and carbohydrates contain the
same amount of usable energy about 4 kcal/g. However, proteins are a very
inefficient source of energy, due to the need for the metabolism and processing by
liver and kidneys, prior to the utilization of this energy source.
1.5
The Basic Proteins Quantification Protocols
Numerous methods for determining the amount of proteins present in a
sample have been devised over the years. The selection of analytical technique must
depend on the definition of protein. Moreover, the criteria for choice of a protein
assays are usually based on convenience, availability of protein for assays, presence
or absence of interfering agents and accuracy. Several indirect ways to measure
protein concentrations spectrophotometrically have been developed [15]. This
method depended on:
i.
Amount of protein
ii.
Concentration of protein
iii.
Presence of compound which may interfere with assay
iv.
Specificity of assay
v.
Ease and reliability, accuracy and precision of performing the assay
1.5.1
Colorimetric Method
Colorimetric assays for proteins are based on certain metal ions and dyes
binding to protein in a specific mass ratio and upon binding become intensely
colored. These reagents were reacted with protein samples to obtain an absorption
band, which the intensity is linearly proportional to the protein concentration of a
9
solution in mass/volume units. The three most commonly used of these methods are:
the Biuret assay, the Lowry assay and Bradford assay [16].
1.5.1.1 Biuret Method
The Biuret reaction is the most specific of the protein methods. The assay
was developed following the observation that Biuret NH2CONHCONH2 reacts with
an alkaline solution of copper sulphate to give a purple coloured complex [17, 18].
The intensity of the charge-transfer absorption band resulting from the copperprotein complex is linearly proportional to the mass of proteins present in solution.
This method is a relatively insensitive method (detection limit ~ 0.5 to 80 mg/mL),
but provides a fast and simple means of obtaining estimates of proteins concentration
over a rather large range of concentration.
1.5.1.2 Lowry Method
Lowry method [19] is a widely used quantitative assay for determining
proteins content in a solution. The method based on both Lowry and Biuret reactions,
where the peptide bonds of proteins react with copper under alkaline conditions
producing Cu+, which reacts with the Folin reagent. Folin-Ciocalteau reaction is
based on phosphomolybdotungstate reduced to heteropolymolybdenum blue by the
copper-catalyzed oxidation of aromatic amino acids [16]. Besides that, the reactions
resulted in a strong blue colour, due to presence of tyrosine and tryptophan in
proteins. Most interfering substances removed by precipitating the proteins from
solution prior to running the assay. The detection range of this method is 1 to 300
µg/mL. Advantages of this method are reliable method for proteins quantitation and
little variation among different proteins. The disadvantages of this method are many
interfering substances, slow reaction rate, instability of certain reagents and proteins
irreversibly denatured.
10
1.5.1.3 Bradford Method
An assay originally described by Bradford [20] in 1976 has become the
preferred method for quantifying proteins in many laboratories. This technique is
simpler, faster and more sensitive than the Lowry method. This assay relies on the
binding of the dye Coomassie Brilliant Blue G250 to proteins [20]. Under the pH
conditions of the assay, the dye is present in its cationic form and does not absorb
strongly at 595 nm. When the dye binds to a protein, there is a stabilization of the
doubly protonated anionic form of the dye, which absorb in the 595 nm region [21].
The dye appeared to bind most readily to arginyl residues of proteins (but does not
bind to the free amino acids) [21]. This specificity can lead to variation in the
response of the assay to different proteins.
The technique of this assay is simple, faster (carried out in a single step) and
more sensitive than the Lowry method. The method is capable of detecting as little as
0.5-50 µg/mL of proteins and the range of sensitivity is 20-200 µg/mL. One of the
major advantages of this assay is insensitive to interferences from reagents that are
commonly found in proteins solutions. Disadvantages of this method are the proteins
used for this assays are irreversibly denatured [22].
1.5.2
Bicinhoninic Acid (BCA) Method
The bicinchoninic acid (BCA) assay was first described by Smith et.al. [23].
This developed method was a modification of the Lowry method. This method also
depends on the conversion of Cu2+ to Cu+ under alkaline condition and the Cu+
detected by reaction with BCA. The two assays are similarly sensitive, but since
BCA is stable under alkali conditions, this assay has the advantage that it can be
carried out as a one-step process compared to the two steps needed in the Lowry
assay. The reaction resulted in the development of an intense purple color with an
absorbance maximum at 526 nm. Since the production of Cu+ in this assay is a
function of proteins concentration and incubation time, the protein content of
unknown samples can be determined by comparison with known protein standards.
11
The advantages of this assay are fewer interfering substances compared to
Lowry assay and it is not affected by range of detergents and denaturing agents. A
further advantage of this method is that it can be carried out as one-step process
compared to the two steps needed in the Lowry method. However, the disadvantages
of this method are some interferes with different proteins; colour is unstable with
time and colour variations with different proteins. Besides, the presence of sulfhydryl
agents [24] and lipids gives excessively high absorbance [25, 26], which interfere
with the assay.
1.5.3
Kjeldahl Method
Johann Kjeldahl developed this Kjeldahl original method in 1883 [3]. The
method has approval from the AOAC (Association of Official Analytical Chemists),
formerly the Association of Official Agricultural Chemist [27]. The Kjeldahl
methods were based on titration of ammonium ion produced by digestion of the
protein with concentrated sulfuric acid [3]. This method is the legal basis for
determining proteins (like N, (NH4)2SO4, NH4+), which measure the total nitrogen,
and converting this value to a protein equivalent. For proteins of unknown amino
acid composition, quantitation was based on approximate 16% of nitrogen content in
protein. The typical range for nitrogen content is about 12 to 18%, although a few
types of proteins fall well outside this range (e.g. protamine has 30% nitrogen) [3].
The reagents for analysis need to be N-free so that there is no interference
substances present. This procedure is not applicable to material containing N-N or NO linkages. Interference of nucleic acid and other N-containing organic compounds
can be removed by trichloroacetic acid (TCA) precipitation of proteins [28]. The
advantages of this method are inexpensive and accurate. However, the disadvantages
of this method are waste of time (normally takes 2 hours for digestion) and corrosive
reagents [3].
12
1.5.4
UV Absorbance Method
The ultraviolet absorption method uses the UV spectrophotometer to measure
the quantity of protein present. Spectroscopic investigations were initially pursued to
take advantages of the absorption of peptide bonds in the far-UV portion of the
spectrum (190-235 nm) [29] and aromatic amino acids in the near-UV (250-350 nm)
[30]. The presence of proteins in a sample can be determined by measuring the
amount of light absorbed at 280 nm (Abs280). However, most proteins absorb in the
UV region of 250-350 nm with maximum absorption at 280 nm. In this method, the
absorption at UV range 280 nm wavelengths occur when the proteins have aromatic
side chains such as tryptophan (Typ), tyrosine (Try) and phenylalanine (Phe). The
range of sensitivity of this range is between 0.2 -2 mg/mL proteins [31]. Besides that,
the peptide bonds of proteins absorb the far UV with maximum absorption at 205235 nm. This absorption method is more sensitive compare to absorbance at 280 nm
since there are many peptide bonds in proteins and the bonds are essentially constant
for all proteins. At this UV range, a concentration of 0.01-0.05 mg/mL proteins can
be determined.
The advantages of this method are this method is time saving because of the
simple procedure and the sample is recoverable. It is also useful for estimation of
proteins before using a more accurate method. Besides that, this method does not
destroy the sample. However, this method also has some disadvantages including the
interference from other chromophores such as detergent, nucleic acids and lipid
droplets.
1.6
Recent Advances in Protein Analysis
The development of novel assays for proteins is a basic requisite in both
clinical and laboratory tests. Therefore, a great number of assays have continuously
been proposed in recent years such as based on spectrophotometric [32, 33], nuclear
magnetic resonance (NMR) [34, 35], resonances capillary electrophoresis [36],
fourier-transform infrared (FT-IR) [37] and Rayleigh light-scattering (RLS) [38].
13
Recent research in proteins determination had focus on spectrophotometric
method. Zhong et al. [32] described the spectrophotometric determination of protein
based
on
the
binding
interaction
of
protein,
molybdenum(VI)
and
dibromohydroxyphenylfluorone (DBHPF). In this study, the DBHPF–Mo(VI)
complex was applied as a new spectroscopic probe for proteins and a novel method
for the determination of proteins were developed in the presence of Triton X-100.
The assay was characterized with high sensitivity, long stability, good selectivity and
simplicity. Besides, the binding of 2-hydroxy-3-nitro-9-fluorenone (HNF) (a new
reagent with absorption antitumour activity) to human serum albumin (HSA) was
investigated by Hong et al. [33]. This study was carried out by fluorescence
spectroscopy combined with UV-Vis, circular dichroism (CD) and Fourier transform
infrared (FT-IR) spectrophotometric techniques under simulative physiological
conditions. Accordingly, the experimental results observed in this work indicated
that the binding of HNF to HSA led to the conformational change of HSA.
Besides, nuclear magnetic resonance (NMR) spectroscopy is a powerful
technique for the study of the structure, dynamics and folding of proteins in solution.
Cui et al. [34] demonstrated that nuclear magnetic resonance (NMR) spectroscopy
could be used as an alternative approach to study the competitive binding of two
ligands ibuprofen (IBP) and salicylic acid (SAL) to protein (HSA) at the low-affinity
binding sites. In this study, they utilized an excess of ligands, IBP and SAL, over the
albumin in order to saturate the high-affinity binding sites and to ensure that the
competitive binding of IBP and SAL to HSA. Here, the competitive binding was
analyzed quantitatively using NMR based
1
H spin-lattice relaxation (R1)
measurements, NMR methods that can be use to study molten globule states of
proteins have been described by Redfield [35]. This study has been illustrated using
D-lactalbumin and apomyoglobin, which is two of the most widely studied molten
globules. In this study, it was reported that the specific method used to study the
molten globule state of a particular protein is determined by the quality of the NMR
spectrum obtained. Accordingly, these NMR methods have provided detailed
information about the specific residues involved in secondary structure within the
molten globule.
14
Moreover, capillary electrophoresis is a powerful separation tool for proteins.
Lee et al. [36] demonstrated a method for the analysis of picomolar concentration
proteins using electrophoretically mediated microanalysis (EMMA) to label proteins
on-column with a fluorogenic reagent. This labeling method was followed by
capillary zone electrophoresis separation and post column detection based on laserinduced fluorescence. Accordingly, this method provides separation efficiency of
300 000 theoretical plates and compared to UV absorbance detection; the EMMA
method provides 7 000 000-fold improvement in detection limit.
Fourier transform infrared (FT-IR) is a powerful technique for the study of
hydrogen bonding and very useful for the structural characterization of proteins. The
most important advantage of FT-IR spectroscopy for biological studies is that spectra
of almost any biological system can be obtained in a wide variety of environments.
There have been many studies carried out to investigate the interaction of proteins by
FT-IR technique. Neault et al. [37] reported the FT-IR spectroscopic characterization
on the interaction of cis-diamminedichloroplatinum (II) (cisplatin), an anticancer
drug with human serum albumin. In this study, spectroscopic evidence regarding the
drug-binding mode, drug binding constant and the protein secondary structure was
provided.
In recent years, a new sensitive technique concerning resonance Rayleigh
light-scattering (RRLS) technique has become a new attractive method for the
determination of trace biomolecules such as protein. Based on that, Gang et al. [38]
demonstrated the determination of proteins based on the interaction with
carboxyarsenazo (CAA) by Rayleigh light scattering (RLS). It was found that the
weak RLS of CAA enhanced greatly by the addition of proteins. Thus, the CAA
assay based on RLS method is useful for routine analytical purposes. However, this
technique traditionally regarded as suffering from the disadvantages of low signal
levels and lack selectivity [38].
15
1.7
Fluorescence Method in Proteins Analysis
Sir G.G. Stokes described the phenomena of the fluorescence (absorption and
emission) process in 1852. Sir G.G.Stokes suggested the name fluorescence is from
the mineral fluorspar (lat fluo, to flow and spar, a rock), which produces a blue white
fluorescence [39, 40]. Fluorescence occurs when a molecule absorbs photons from
the UV-visible light spectrum (200-900 nm) and causing transition to a high-energy
electronic state and then emits photons as it returns to its initial state, in less than 10-9
sec [39]. In fluorescence studies, one is generally concerned with two types of
spectrum, the excitation spectrum and emission spectrum. The excitation spectrum is
determined by measuring the emission intensity at a fixed wavelength while varying
the excitation wavelength. The emission spectrum is determined by measuring the
variation in emission intensity wavelength for the fixed excitation wavelength. The
difference between the excitation and emission wavelengths is called as Stoke’s shift.
Fluorescence has proven to be a versatile tool for a myriad of applications. It
was a powerful technique for investigation of molecular interactions in analytical
chemistry, biochemistry, cell biology, physiology, nephrology, cardiology,
photochemistry and environmental science. In the last twenty years, fluorescence
spectroscopy has evolved into a powerful tool for the studies of chemical,
semiconductor, photochemical and biochemical species [40].
Fluorescence spectroscopy has become widely accepted as a modern method
for the study of proteins structure, proteins biosynthesis and other biochemical
problems. The characteristics of proteins fluorescence are dependent on structure of
protein. The fluorescence of peptides is caused by the presence of the amino acids
tyrosine, tryptophan and phenylalanine [41]. However, the fluorescence of
phenylalanine only observed in the absences of tyrosine and tryptophan due the small
quantum yield. Although, tyrosine is a weaker emitter than tryptophan, but it still
contribute significantly to protein fluorescence because it is usually present in larger
numbers. The fluorescence of tyrosine can be easily quenched by nearby tryptophan
residues due to the energy transfer effect. Hence, generally only fluorescence of
tryptophan was detected although in proteins where both tyrosine and tryptophan are
present [41, 42]. Some proteins may also contain a fluorescent coenzyme such as
16
reduced nicotinamide-adenine dinucleotide, flavin-adenine dinucleotide or pyridoxal
phosphate. Proteins in complex with other fluorescent molecules can be term as an
extrinsic fluorescence [42].
Various studies of determining protein have previously been reported in the
literature [43, 44, 45]. Cui et al. [43] demonstrated the interactions between 1benzoyl-4-p-chlorphenyl thiosemicarbazide (BCPT) and bovine serum albumin
(BSA) or human serum albumin (HSA) by fluorescence spectroscopy. Accordingly,
the analysis of fluorescence spectrum and fluorescence intensity showed that BCPT
has a strong ability to quench the intrinsic fluorescence of both bovine serum
albumin and human serum albumin through a static quenching procedure.
Wiberg et al. [44] have developed a method for the simultaneous
determination of albumin and immunoglobulin G (IgG1) with fluorescence
spectroscopy. In this study, few parameters such as excitation and emission slit,
detection voltage and scan rate were investigated. Accordingly, the proposed method
is fast and required a minimum of sample pre-treatment and further development of
the method might make the possibility to determine albumin and IgG directly in
human serum. Besides, the study of the interactions of BSA and HSA with anionic,
cationic and zwitterionic surfactant monitored by fluorescence spectroscopy was
demonstrated by Gelamo et al. [45]. The result showed that the fluorescence of the
interaction of BSA and the surfactant was quenched and the interaction of surfactants
with HSA enhanced the fluorescence, which was an opposite effect compared to
BSA.
1.7.1
Luminescent Organic Compounds Labeling Proteins
Labeling of biological molecules using fluorescent compounds is a common
and very useful practice in biological science and biomedical science. The method of
labeling proteins was introduced by Coon in 1941 when they demonstrated the use of
antibody labeling with fluorescein [42]. This research tool has been proven useful in
17
immuno-chemistry,
virology,
bacteriology,
parasitology,
rickettsiology
and
mycology.
Fluorescent of small molecules such as organic dyes are used in both single
and multiplex detection approaches. The changes in the fluorescence properties of
dyes adsorbed or covalently attached to macromolecules may change the signal in
the microenvironment of the dyes due to configurational changes in the larger
molecule [40]. Reviews on the application of fluorescent compound covalently
bounded with protein have been reported by several studies. Jiang et al. [46] reported
the studies of the binding reaction and the effect of the energy transfer between
terazosin and bovine serum albumin (BSA) by spectrofluorimetry. In this study, it
was reported that the compound formed between terazosin and BSA quenched the
fluorescence of BSA and the synchronous fluorescence technique was successfully
applied to determine terazosin in blood serum and urine samples under physiological
conditions.
Besides, a new method based on fluorescence quenching, employing a red
region fluorescent dye, tetra-substituted sulphonated aluminium phthalocyanine
(AlS4Pc) for the determination of albumin and globulin without separation was
presented by Li et al. [47]. It was reported that, this experimental method was easy
and time saving method and a red-region fluorescent dye with large Stoke’s shift was
employed to minimize the background signal of samples. Moreover, the fluorescent
dye was easily synthesized and chemically stable which makes this method suitable
for practical application.
Jiang and Li [48] have demonstrated the approach of a new fluorescence
method for the determination of human serum albumin (HSA) by using doxycycline
(DC)-europium (Eu3+) as fluorescence probe. Accordingly, HSA remarkably
enhanced the fluorescence intensity of the DC-Eu3+ complex and the enhanced
fluorescence intensity of DC-Eu3+ is proportional to the concentration of HSA.
Ercelen et al. [49] reported on highly specific and stochiometric binding of novel
fluorescence probe FA, 2-(6-diethlaminobenzo[b]furan-2-yl)-3-hydroxychromone to
bovine serum albumin (BSA).
18
Chun et al. [50] demonstrated Erythrosin B (EB) binding to proteins, which
caused a decrease in the fluorescence maximum of EB. In this study, fast and simple
fluorescence quenching method for the determination of proteins was developed.
Accordingly, the method has advantages in aspect of high sensitivity, short reaction
time, good reproducibility, stable fluorescence and very little interference developed
simple, rapid and sensitive fluorescence method for determining microamount
albumin. The same group demonstrated a simple, rapid and sensitive fluorescence
method for determining microamount albumin [51]. According to the report, the
fluorescence of 2, 5-di (orthoamino phenyl)-1, 3, 4-oxadiazole is enhanced greatly by
the addition of albumin and little interferences on the assay were determined.
Besides, a new form of highly luminescent and photostable particles was
generated by doping the fluorescent dye tris-(2ƍ2-bipyridyl) dichlororuthenium(II)
hexahydrate (Rubpy) inside silica material was successfully employed in various
fluorescence labeling by Wei et al. [52]. This study demonstrated the potential to
apply these newly developed fluorescent particles in various bio-detection systems.
Perez-Ruiz et al. [53] demonstrated similar fluorescence quenching method based on
binding of the dye. The study involved the quantitative analysis of protein based on
the lower fluorescence of Rose Bengal dye binding to the proteins. This approach
was satisfactorily applied to the determination of total proteins in different serum
samples.
Shobini et al. [54] have published a comprehensive review of the interactions
of 7-aminocoumarins with human serum albumin (HSA) by using fluorescence
spectroscopic technique and modeling studies. In this study, a large change in
fluorescence spectral parameters such as intensity, emission maxima and anisotropy
for aminocoumarins was observed.
Although most of the reported protein assays are based on the binding of the
dyes or organic compounds with protein has been proven the most sensitive assay
and very useful practice in biological science and biomedical science, however this
organic fluorophores have characteristics that limit their effectiveness for such
applications. These limitations included narrow excitation bands and broad emission
bands with red spectral tails, which can cause simultaneous evaluation of several
19
light-emitting probes problematic due to spectral overlap [55]. Moreover, the organic
fluorophores often suffer from photobleaching, low signal intensities and random
on/off light emission (blinking) [56]. Photobleaching were caused by sudden
decomposition of the emitter, which the main factor limiting the maximum number
of photons obtained from a fluorophores. Besides that, low signal intensities reduces
the accuracy of the trace protein can be determined. Intermittent light emission
causes problems in real-time studies of biomolecular dynamics such as protein
folding, signal transduction and enzymatic catalysis [56].
Due to this, findings strongly suggested that colloidal semiconductor have the
potential to overcome problems encountered by organic fluorophores in certain
fluorescent labeling application by combining the advantages of high photobleaching
threshold, good chemical stability and readily tunable spectral properties. Besides
that, their resistance to photobleaching and high quantum yield in aqueous solution
make them attractive for labeling functionalized biomolecules for fluorescent
applications [55, 56, 57].
1.7.2
Luminescent Semiconductor Labeling Biomolecules.
Semiconductor particles are often composed of atoms from groups II-VI or
III-V elements in the periodic table. Extensive research in the past 20 years has
focused on the photophysics of nanostructures and their applications in
microelectronics and optoelectronics [58, 59]. However, recent developments
indicate that the first practical applications of semiconductor are occurring in biology
and medicine [30, 31]. Semiconductor particles are considerable current interest, not
only because of their unique size-dependent properties but also because of their
dimensional similarities with biological macromolecules such as nucleic acids and
proteins [60].
Recent advances in materials research have produced a new class of
fluorescent labels by conjugating semiconductor with biorecognition molecules.
These fluorophores will have key applications in biotechnology and bioengineering.
20
This review focuses on the biological applications of semi-conductor colloidal
particles. Mattoussi et al. [60] described a novel and direct method for conjugating
protein molecules to luminescent CdSe-ZnS core-shell for use as bioactive
fluorescent probes in sensing, imaging, immunoassay and other diagnostics
applications. In this study, the design and preparation of a semiconductor and protein
conjugate based on E. coli Maltose binding protein was introduced and followed by
functional characterization using luminescence method. Accordingly, the preparation
of protein modified semiconductor dispersions with high quantum yield, little or no
particle aggregation and retention of biological activity was achieved based on
fluorescence method.
Gerion et al. [61] described the synthesis of water-soluble semiconductor
particles, discussed and characterized their properties. The study involved the
hydrophobic CdSe and ZnS were embedded in a siloxane shell and functionalized
with thiols and amine groups. Accordingly, the introduction of functionalized groups
into the siloxane surface would permit the conjugation of the semiconductor to
biological entities.
Parak et al. [62] demonstrated a water-soluble and highly fluorescent
silanized semiconductor nanocrystals were covalently attached to biological
macromolecules with a variety of mild coupling chemistries. In this study, siloxane
shells derivatized with thiols, amino and carboxyl functional groups was coupled to
single or double stranded DNA. The author made a conclusion that, by using the
strategies developed in this study, most biomolecules can be covalently coupled to
semiconductor nanocrystals. Further, these nanocrystal-DNA conjugates promise to
be a versatile tool for fluorescence imaging and probing of biological systems.
1.7.2.1 Cadmium Sulfide Labeling Biomolecules
Cadmium Sulfide (CdS) is an important semiconductor owing to its unique
electronic, optical properties and its potential application in solar energy conversion,
non-linear optical, photo electrochemical cells and heterogeneous photo catalysis
21
[63]. Cadmium sulfide occurs in nature as the mineral greenoktite. The physical
properties are yellow to orange crystal. CdS occurs as two polymorphs, hexagonal
alpha form and cubic beta form. It exhibits stable wurtzite structure at lower
temperature and zinc blended type structure at the higher temperature [64]. CdS
compound was widely used in pigments for paints, baking enamels is ceramics
plastics. Other applications of this compound are in photovoltaic solar and cells (for
converting solar energy to electrical energy), photoconductors (in xerography), thin
film transistor and diodes, rectifiers, scintillation counters pyrotechnics and smoke
detectors [64].
The synthesis of CdS has been tried by various methods such as the direct
reaction of metals with sulfur powder under high temperature, the thermal
decomposition of molecular precursors containing M-S bonds and chemical
precipitation method involving the precipitation of metal ion with Na2S as the source
of S2- ions [64]. The type of caping agents is a great importance in synthesis CdS
since it affects the chemical as well as the physical properties of the semiconductor
in term of stability and solubility [65, 66]. Most commonly used stabilizers are
pyridine, alkyl amines and various thiols [66, 67, 68].
Recent research by several groups has linked colloidal CdS particles to biomolecules. Mahtab et al. [68] have examined the adsorption of different DNA
sequences to mercaptoethanol-capped CdS quantum dots as assay for nonspecific
protein DNA interactions. In this study, the binding constant for mercaptoethanolcapped CdS with protein DNA interactions was established. Besides, the adsorption
of calf thymus DNA to particles of Cd(II)-rich CdS has been examined by
photoluminescence spectroscopy as a function of temperature. Moreover, Mathab et
al. [69] also performed the adsorption of calf thymus DNA to Cd(II)-rich CdS
particles by photoluminescence spectroscopy as a function of temperature. This study
suggested that the driving force for adsorption is entropy, and the enthalpy
contribution to DNA-surface binding is slightly unfavorable. Similar results also
reported by Lakowicz et al. [70] who studied about time–resolved fluorescence
spectra of cadmium-enriched particles (CdS-Cd2+) and the effect of DNA oligomer
binding.
22
Wang et al. [71] demonstrated a study of a novel composite particle prepared
by an in situ polymerization method and applied as a protein fluorescence probe. In
this study, the CdS has been prepared by polymerization with acrylic acid (AA) and
the surface of the composite CdS particles was covered with abundant carboxylic
groups (–COOH) to link proteins. Li and Du [72] have studied the synthesis CMCH
(carboxymethly chitosan)-capped CdS particles. According to the report, this
polysaccharide (carboxymethly chitosan)-capped CdS would have more affinity with
other biomolecules such as protein and DNA molecules, thus making it a promising
fluorescent bio-label.
1.7.2.2 Zinc Sulfide Labeling Biomolecules
Zinc sulfide (ZnS) is one of the II-VI semiconductor compounds, which have
wide ranging applications in solar cells, infrared window materials, photodiode and
cathode-ray tube [73], electro luminescent devices [74] and multiplayer dielectric
filters [75]. ZnS occurs in two crystalline forms, one in the hexagonal system and
other in cubic system. The minerals in hexagonal system are called as wurtzite
whereas the cubic system known as sphalerite or zinc blended. Zinc sulfide is
insoluble in water and the physical properties of ZnS are white to gray-white or pale
yellow powder [64].
Considerable progress has been made in the synthesis of zinc sulfide
powders. Irradiation method was extensively used to prepare the ZnS particles were
introduced by Qiao et al. [76]. In this method, different sulfur sources such as
sodium thiosulfate, thiourea and mercaptoethanol were used successfully to obtain
ZnS. Due to the complexity and expensiveness of some of these methods, Wang et
al. [77] developed a new solid-state method by which zinc sulfide particles can be
obtained easily via solid-state chemical reaction of zinc acetate and thioacetamide at
low temperature. However, all the methods described above are difficult to
commercialize due to the high-cost and low-volume capacity. Finally, Kho et al. [78]
proposed the most acceptable method for the ZnS preparation. This approach
23
described a simple, inexpensive and reproducible procedure for large-scale synthesis
of highly stable of ZnS capped cysteine particles.
Several research groups have reported few reviews on the ZnS semiconductor
linked biomolecules. Chan et al. [79] presented a highly luminescent semiconductor
(zinc sulfide-capped cadmium selenide) have been covalently coupled to protein for
ultra-sensitive biological detection. According to the report, this class of luminescent
labels is 20 times as bright, 100 times as stable against photobleaching and one-third
as wide in spectral line width compared with organic dyes such as rhodamine.
Besides, these semiconductor conjugated with biomolecules are water-soluble and
biocompatible.
Wang et al. [80] presented a more detailed study on application of ZnS watersoluble particles modified with sodium thioglycolate. In this study, the modified ZnS
were used as fluorescence probes for the quantitation determination of proteins and
was proved a simple, rapid and specific method. It has been concluded that these
semiconductor particle probes are brighter, more stable against photobleaching and
do not suffer from blinking compared with single organic fluorophores.
Li et al. [81] presented water-soluble cysteine capped ZnS particles as
fluorescence probe for the determination of DNA. In this study, a synchronous
fluorescence method has been developed for the rapid quantitative determination of
DNA with ZnS capped cysteine.
1.8
Advantages and Disadvantages of Fluorescence in Proteins Analysis.
Fluorescence is a particularly important analytical technique because of its
extreme sensitivity and good specificity.
1.
First there are two wavelengths used in fluorescence analysis. Emitted light
from each fluorescent color can be easily separated because each color has
unique and narrow excitation spectra. Besides that, multiple fluorescent
24
colors within a single sample can be quantified by sequential measurement of
emitted intensity using a set of excitation and emission wavelength pairs
specific for each color.
2.
Second advantage of fluorescence is low signal to noise ratio because the
emitted light enters the emission monochromator, which is positioned at 90o
angels from the excitation light path to eliminate background signal and
minimize noise due to stray light.
3.
The third advantage is that fluorescent methods have a greater range of
linearity. Due to this, the sensitivity of fluorescence is approximately 1000
times greater than absorption spectrometric methods.
1.8.1
Disadvantages
1.
Fluorescence is particularly sensitive to contaminating substances. Thus, for
example, the cuvette may be contaminated by fluorescence material.
2.
The fluorescence reading is not stable due to variety of reasons below.
i.
Fogging of the cuvette when the contents are much colder than the ambient
temperature.
ii.
Bubbles forming in the solution.
iii.
Drops of liquid on the external faces of the cuvette.
25
1.9
Research Background
The development of novel assays for proteins is a basic requisite in both
clinical and laboratory tests. The analysis of protein can be used as a reference for
measurements of other components in biological fluids and clinical diagnosis. The
most frequently used approaches for the determination of protein is the ultraviolet
and visible absorption spectroscopy, Lowry method [19], dye binding method like
Bradford [20], Bromocresol green procedures [82] and Bromophenol blue [83].
However, they all have some limitation in terms of sensitivity, selectivity, stability
and simplicity. Disadvantages of the Lowry method include low sensitivity, poor
selectivity and complexity [19]. The Bradford method is also inconvenient in
operation and application due to the requirement for calibration and to the
nonlinearity between the absorbance of the Coomassie Brilliant Blue G-250 (CBB G250) dye-protein complex and the concentration of protein [20]. The bromocresol
green method is insensitive and susceptible to interference by turbidity [82] and the
Bromophenol blue method can be used only for protein concentrations greater than
10 mg L-1 [83]. Therefore, a number of assays have been continuously reported in
recent years such as those based on spectrophotometric [32, 33], nuclear magnetic
resonance (NMR) [34, 35], capillary electrophoresis [36], Fourier transform infrared
(FT-IR) [37] and resonance Rayleigh light-scattering (RRLS) [38].
Findings strongly suggested that, the most sensitive quantitation of protein at
this present is generally based on their fluorescence enhancement effect on organic
dyes. Fluorescent small molecules (organic dyes) are used in multiplex detection
approaches that had achieved a considerable level of sophistication with character of
rapidity, good selectivity and high sensitivity. However, the organic fluorophores
often suffer from photo bleaching, low signal intensities, random on/off light
emission (blinking) and narrow excitation [55, 56].
Colloidal semiconductor particles have the potential to overcome problems
encountered by organic small molecules in certain fluorescent labeling applications
by combining the advantages of high photobleaching threshold, good chemical
stability and readily tunable spectral properties [55]. These colloidal particles are
very resistance to photobleaching and have a high quantum yield in aqueous solution
26
make them attractive for labeling functionalized biomolecules for fluorescent probe
[55]. In this study, the new direction for the synthesis of CdS capped with mercapto
acetic acid and ZnS capped with cysteine was employed for the binding with protein.
This functionalized CdS and ZnS will be covalently link to protein (BSA) for further
studies.
1.10
Research Objective and Scope of Study
Fluorescence method is one of the most sensitive methods frequently used in
the determination of protein. The interest in the use of fluorescence technique of
determination involves the development of methods that are faster, more efficient,
accurate, simple and more sensitive for the determination of protein. Recent
advances in fluorescence studies have produced a new class of fluorescent labels by
binding semiconductor with biological compounds.
The objective of this study is to:
i.
Synthesize the CdS capped with mercaptoacetic acid and ZnS capped
with cysteine.
ii.
Develop the method for Bovine serum albumin binding with
functionalized CdS and functionalized ZnS.
iii.
Apply the developed method in the quantitative analysis of protein
In this study, functionalized CdS and functionalized ZnS binding with protein
(BSA) was characterized with fluorescence technique. The binding fluorescence
properties of functionalized CdS and ZnS with BSA were optimized for the
quantitative analysis of protein. The effect of interfering substances and surfactant
binding with functionalized colloidal and BSA was performed. In order to employ
the developed method, different type of protein such as albumin, lysozyme and
amylase detection was carried out.
CHAPTER 2
EXPERIMENTAL
2.1
Apparatus
Perkin-Elmer Models LS-50B Luminescence Spectrometer was used for all
fluorescence measurements (Figure 2.1). The LS-50B Luminescence Spectroscopy
employed a pulse xenon source that produce a high output using a low voltage, 9.9
watts resulting in longer lamp life with minimal ozone and heat production.
Photomultiplier tubes were employed as detection devices and LS-50B was
connected to a computer for data processing. A Shimadzu UV-1601PC UV-visible
spectrophotometer was used for UV-visible spectrometry analysis. A pH meter
Cyber-scan model equipped with a glass electrode combined with an Ag/AgCl
reference electrode, was employed for the pH measurements.
28
Figure 2.1: Luminescence spectrometer Perkin-Elmer Model LS-50B
2.2
Reagents
All the glassware used in this experimental work was acid washed. The
chemical reagents used were analytical reagent grade without further purification.
Water, purified in a Nano Pure Ultrapure water system (Barnstead /Thermolye) was
used for all dilution and sample preparation. Cadmium chloride (CdCl2.H2O), sodium
hexametaphosphate (NaPO3)6 and sodium sulfide (Na2S x.H2O) were obtained from
Ajax Chemicals (Australia). L-cysteine, L-glycine and mercaptoacetic acid were
purchased from Merck (Germany) whereas L-alanine, L-tryptophan and hydrochloric
acid were purchased from Fluka (Switzerland). Samples of Bovine Serum Albumin
(BSA), albumin, lysozyme, amylase and tris buffer were obtained from Amersham
Pharmacia Biotech (U.S.A). SDS was obtained from BDH Chemicals (England)
while Triton-X was purchased from Sigma Chemical U.S.A.
29
2.3
Preparation of Standard Solutions
2.3.1
Preparation of 0.1 mol L-1Cadmium Chloride (CdCl2.H2O)
Cadmium chloride (CdCl2.H2O) (2.013 g) was dissolved in 100 mL
volumetric flask using distilled water.
Preparation of 0.1 mol L-1 Sodium Sulphide (Na2S.9H2O)
2.3.2
Sodium sulfide (8.05 g) was dissolved in 100 mL volumetric flask of distilled
water until the final concentration of Na2S.9H2O is 0.1 mol L-1. The solution was
diluted with distilled water to the mark.
Preparation of 1.0 mol L-1 Zinc Sulphate (ZnSO4)
2.3.3
Zinc sulfate (7.189 g) was dissolved in 25 mL volumetric flask of HCl 0.01
-1
mol L . Then the solution makes up to the mark and shakes well.
2.3.4 Preparation of 1.0 mol L-1 Tris buffer (tris hydroxymethyl amino
methane)
Tris buffer (tris hydroxymethyl amino methane) (6.057 g) was dissolved into
a 50 mL volumetric flask using distilled water and make up the solution to the mark.
30
2.3.5
Preparation of Hydrochloric Acid (HCl) 0.01 mol L-1
Hydrochloric acid (0.01 mol L-1) was prepared by adding 0.041 mL of
hydrochloric acid 12.23 mol L-1 into a 50 mL volumetric flask with distilled water.
2.3.6
Preparation of Mercaptoacetic Acid 1.0 mol L-1
For the preparation of 1.0 mol L-1 of mercaptoacetic acid, 0.709 mL of
mercaptoacetic acid was diluted in 10 mL of volumetric flask with distilled water to
the mark.
2.4
Preparation of Stock Solutions
2.4.1 Preparation of Bovine Serum Albumin (BSA), Albumin, Lysozyme,
Amylase Stock Solution (100 ppm)
Bovine Serum Albumin was prepared by directly dissolving 0.01 g of BSA in
0.5% NaCl solution in 100 mL volumetric flask. The final concentration of BSA 100
ppm was stored at 0-5 oC. The same procedure was carried for the preparation of 100
ppm of albumin, lysozyme and amylase stock solution.
2.5
Procedure for Preparing CdS Capped Mercaptoacetic Acids
The basic CdS colloids were prepared following a method described by
Spanhel et al. [84]. The procedure employed for preparing CdS particles is as follow.
The synthesis of colloidal solution was carried out in a 1000 mL a round bottom
flask. Then, 800 mL of deionized water, 4.0 mL of 0.1 mol L-1 CdCl2.H2O and 4.0
mL of 0.1 mol L-1 sodium hexametaphosphate as the stabilizing agent were added to
the flask. Then, the pH was adjusted to 7.0 using 0.1 mol L-1 NaOH. Under vigorous
31
stirring, 4.0 mL of 0.1 mol L-1 Na2S.9H2O, was dropped into flask slowly. Then, 1.0
mL of 1.0 mol L-1 of mercaptoacetic acid was reacted with mercaptoacetic acid for 3
hours under vigorous stirring. Finally, the excess mercaptoacetic acid was removed
by repeated centrifugation. The functionalized colloidal solution was stored at the
room temperature.
2.6
Procedure for Preparing ZnS Capped Cysteine
The simple procedure of synthesis ZnS capped cysteine was described by
Kho et al. [78]. 1.970 g of L-cysteine was added to 50 mL of degassed and N2
saturated 1.0 mol L-1 Tris buffer (tris hydroxymethyl amino methane). ZnSO4 (1.0
mol L-1, 6.25 mL) was then added to the solution. Then, 25.0 mL of 0.4 mol L-1
Na2S.9H2O was added dropwise into the solution slowly followed by stirring the
mixed solution stirred for 30 min. Finally, the colloidal solution was sealed and
incubated at 47 oC bath water, followed by 10 min of purged N2 to remove the excess
sulfide. The purified ZnS capped cysteine powders were obtained through ethanol
precipitation. In this procedure, 100% cold ethanol (0 oC) was added drop wise in the
presence of about 10% (v/v) 1.0 mol L-1 NaC2H3O2 (sodium acetate) until a white
precipitate ZnS was formed. After the centrifugation for 15 min, the pellet was
redissolved in 1.0 mol L-1 Tris buffer. At least two rounds of ethanol precipitation
were necessary to remove most of the excess components. To produce the final
powder, the ethanol precipitated and redissolved sample was dried overnight. Finally,
these powder products were stored at 4 oC.
2.7
Fluorimetric Analysis
Emission and excitation spectra of all the solution were obtained using
luminescence spectrometer. Fluorescence instrument parameters were set up prior to
analysis. All the spectra were measured using 5 cm quartz cell and scanned with a
fixed scan speed (250 nm/min). The emission and excitation spectra for
32
functionalized CdS binding with BSA were obtained using 8 nm slit-widths while for
functionalized ZnS binding with BSA the slit-width was setup at 5 nm.
Into a 5 ml volumetric flask, 100 ȝL of buffer solution, 1500 ȝL of
functionalized CdS and appropriate volume of protein sample were added and diluted
to 5.0 mL of distilled water. Then, fluorescence excitation spectrum was recorded
from 200 nm to 600 nm and fluorescence emission spectrum was scanned with a
fixed excitation at Ȝex = 233 nm. Wavelength of emission spectrum at Ȝem = 350 nm
and the intensity of maximum peaks were recorded.
2.8
UV-Vis Spectroscopy Analysis
The prepared solutions of functionalized CdS in presence of various
concentration of BSA were analyzed using UV-visible spectrophotometer in the
range of 200 nm to 800 nm. Absorbance at 280 nm wavelength was recorded.
Calibration curve of absorbance at specific wavelength against standard solution
concentrations was plotted.
2.9
General Procedures
2.9.1
Effect of pH
The functionalized CdS colloidal was prepared in various pH values such as
pH 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11 using Tris buffer. Next, the fluorescence emission
spectra was measured when the excitation spectra was at 233 nm wavelength.
Further, the emission spectra in presence of 25 ppm protein (BSA) were measured
for various pH. The same procedures were carried out for the measurement of
functionalized ZnS colloidal in presence of 15 ppm of BSA.
33
2.9.2
Effect of Buffer
Experimental procedures demonstrated with three different kinds of buffers in
functionalized CdS colloidal in absence and presence of 25 ppm of BSA at optimum
pH. The three buffers were Tris-HCl, BRB-NaOH and NaAc-HCl. The fluorescence
emission spectra at Ȝem=350 nm for each buffer were measured. The same procedures
were repeated for the measurement of functionalized ZnS colloidal in presence of 15
ppm of BSA.
2.9.3
Effect of Concentration
The effect of functionalized CdS concentration on the fluorescence intensity
was performed by measuring the emission intensity of functionalized CdS
concentration from 1x10-4 mol L-1 to 4.5x10-4 mol L-1 at the selected optimum pH.
The emission spectra at Ȝem = 350 nm wavelength were measured until the
concentration of the colloidal solution reached the maximum value. These
procedures were repeated for the measurement of functionalized ZnS colloidal in
range 1x10-4 mol L-1 until 8x10-4 mol L-1 at optimum pH.
2.9.4 Effect of Temperature
The fluorescence intensity of CdS colloidal was analyzed at different
temperatures ranging from 10 0C to 40 0C in the absence and presence of 25 ppm of
BSA Then the emission spectra were measured at Ȝex = 233 nm under optimum pH.
These procedures were repeated for the measurement of functionalized ZnS colloidal
in the absence and presence of BSA of 15 ppm.
34
2.9.5
Effect of Reaction Time
The optimum reaction time of CdS colloidal in the presence of 25 ppm of
BSA was obtained by measuring the fluorescence intensity at 350 nm for 3 hours
period immediately after mixing. The mixture was measured every 5 minutes for 3
hours period. These experimental procedures were carried for the mixture of
functionalized ZnS binding with BSA 15 ppm at optimum pH and temperature.
2.9.6
Calibration Curves for the Quantitative Analysis of BSA
This procedure was carried by measuring the fluorescence intensity of
functionalized CdS in the presence of various concentration of BSA under the
optimum pH and temperature. A graph of fluorescence intensity versus concentration
was plotted and the calculation for the regression equation and R2 were made using
Microsoft Excel Version 2000. This method was employed for the determination of
functionalized ZnS binding with BSA.
2.9.7
Accuracy of the Method
For the intra-assay determination of accuracy of the developed method, the
reproducibility was calculated from 10 independent runs of functionalized colloidal
and 25 ppm BSA solution on the same day. For inter day accuracy, proposed method
was determined in concentration range of BSA 25 ppm, by six measurements on
different days within three days of different CdS binding with BSA solutions. The
mean, standard deviation and relative standard deviation (RSD) were calculated. The
same accuracy determination procedure was proposed for the functionalized ZnS
binding with 15 ppm BSA under optimum parameter.
35
2.9.8
Limit of the Detection and Quantification
The limit of the detection of the method was obtained using a lower BSA
concentration and at smaller increment additions to the functionalized CdS solution.
The studies were conducted under the optimal condition. The studies on analyte
(BSA) concentration of 0.2 ppm to 4 ppm were conducted under the optimal
condition. Besides, the 3ı of the limit of detection was calculated from 10 blank
measurements of functionalized CdS to obtain low limit of detection value. This
method was employed for the determination of functionalized ZnS binding with
BSA.
2.9.9
Salt–Dependent Studies on Functionalized CdS Binding with BSA
The effect of the ionic strength on the fluorescence emission intensity of
functionalized CdS binding with BSA was investigated by addition of NaCl from 0.1
mol L-1 to 0.5 mol L-1 in the absence and presence of BSA 25 ppm. Graph
concentration of NaCl (mol L-1) versus fluorescence intensity were plotted to
distinguish the ionic strength of functionalized CdS binding with BSA. The
experimental approach was adopted to investigate the ionic strength between
functionalized ZnS binding with BSA 15 ppm.
2.9.10 Effect of Foreign Substances
The experimental procedure for the determination of foreign substances was
obtained under optimum parameter. Various concentrations of few substances such
as metals, non-protein substances, glucose, starch, sucrose and amino acids were
measured at 350 nm wavelength in the presence functionalized CdS binding with
BSA 25 ppm. Same procedure was carried out to obtain the effect of foreign
substances from the determination of functionalized ZnS binding with BSA 15 ppm.
36
2.9.11 Effect of Surfactant
The effects of surfactants such as sodium dodecyl sulfate (SDS), nonionic
detergent Triton X-100 on ZnS binding with BSA emission peak were studied under
the optimum condition. Aliquots of various concentration of surfactant were added to
solution of functionalized CdS binding with BSA 25 ppm. Then, the emission spectra
of the solutions were measured at Ȝem = 350 nm wavelengths. These procedures were
carried for the functionalized ZnS binding with BSA 15 ppm under the optimum
parameter.
CHAPTER 3
FUNCTIONALIZED CADMIUM SULFIDE BINDING WITH BSA
3.1
Preamble
The quantitative analysis of protein is considerably essential in biochemistry
and clinical medicine. Currently, most of the widely used protein assays are
spectrophotometric methods such as Lowry, Bromophenol blue, Bradford and
Bromocresol green. However, they all have some limitation in terms of sensitivity,
selectivity, stability and simplicity [19, 22, 82, 83]. The most sensitive quantitation
of protein at present is generally based on their fluorescence detection [55, 56]. In
this study, a novel luminescent cadmium sulfide (CdS) particle has been employed
for the quantitative determination of protein.
38
3.2
Fundamental
Studies
of
CdS
Capped
Mercaptoacetic
Acid
(Functionalized CdS) Particles
3.2.1
Formation of CdS Capped with Mercaptoacetic Acid (Functionalized
CdS) Particles
The CdS particle formed via the following reaction:
Room
Temperature
CdCl2.H2O + Na2S.9H2O
CdS + 2NaCl + 10H2O
[85]
During the synthesis of CdS particles, it was observed that one deep yellow
colloidal product was formed immediately upon mixing the two reactants. This
phenomenon indicated that the chemical reaction rate of the reactive system is very
fast [85]. Therefore, this reactive chemical reaction favored the formation of small
size CdS particles [85]. Besides, the capping agent mercaptoacetic acid was used as
stabilizer. Further, thiols group from mercaptoacetic acid binds to a Cd atom and the
polar carboxyl group is available for covalent coupling to various biomolecules such
protein and nucleic by cross-linking to active amine group [68, 86].
Figure 3.1: Yellow CdS solution capped with mercaptoacetic acid
39
3.2.2
Fluorescence Properties of CdS Capped with Mercaptoacetic Acid
(Functionalized CdS)
Fluorescent molecules have two characteristic spectra, the excitation
spectrum (the relative efficiency of different wavelengths of exciting radiation to
cause fluorescence) and the emission spectrum (the relative intensity of radiation
emitted at various wavelengths). The emission always occurs at longer wavelengths
than the excitation, which caused by a small energy loss in the brief period before
emission [40].
Fluorescence spectroscopy has been used to characterize CdS capped
mercaptoacetic acid at room temperature. Figure 3.2 shows the excitation spectrum at
233 nm wavelength and emission spectrum of functionalized CdS obtained at 350
nm wavelengths when the excitation wavelength selected from the short wavelength
region at 233 nm wavelengths. The energy difference between the maximum of the
excitation wavelength and the emission wavelength is called the Stoke’s shift. In this
study, the large value of the Stoke’s shift 117 nm was obtained and this may
contribute to the sensitivity of the method. The slit width for all measurement of CdS
capped mercaptoacetic acid was setup at 8 nm. The slit width should be set to high
level in order to get a high intensity of the fluorescence and to avoid background
noise [87].
According to Nagamua et al. [88] emission fluorescence properties of CdS
obtained are more likely due to electron-hole pair radiative recombination from
shallow trap states than from the band edges. The fluorescence emission is due to
recombination of charge carriers via surface traps having energy levels in the
forbidden band gap. This conclusion was in good agreement with that reported by
Spanhel et al. [84].
40
Figure 3.2: Fluorescence (a) excitation spectrum and (b) emission spectrum for CdS
capped with mercaptoacetic acid. Ȝexc = 233 nm, [Functionalized CdS colloids] =
3 x10-4 mol L-1.
3.3
Fluorescence Properties of Functionalized CdS Binding with BSA
3.3.1
Standard Calibration of Absorbance Properties on Functionalized CdS
Binding with BSA
The UV-visible spectroscopy method was used to characterize the binding
between functionalized CdS and BSA. Absorption in the UV range is caused by ʌelectrons, which reflects aromatic and carboxylic electron system [89]. The
absorption spectra of functionalized CdS in the presence of various concentration of
BSA are shown in Figure 3.3. It was observed that, functionalized CdS exhibited
nearly no absorption in the selected region in the absence of BSA. However, the
functionalized CdS showed a slight increase with increasing amount of BSA at
absorption maximum at 280 nm wavelengths. Literature data confirmed the result
obtained [31]. The phenomena occurred is due to the proteins absorbed in the UV
region of 250 nm-350 nm with maximum absorption at 280 nm. Besides, proteins
41
that contained active aromatic amino acids such as tryptophan (Trp) and tyrosine
(Try) have their absorbance maxima between 278 nm and 280 nm [31].
Based on the absorption intensity, the calibration curves of functionalized
CdS in the presence of various concentration of BSA were constructed and it yielded
a linear relationship between the absorbance at 280 nm and BSA substances
concentration in the range 0 to 35 ppm (Figure 3.4). Good correlation with R2 value
of 0.9756 with regression equation y = 0.001x + 0.005 were obtained between UV
absorbance at 280 nm.
Figure 3.3: UV absorbance spectra of functionalized CdS binding with BSA with
increasing concentration 0 ppm to 35 ppm. [Functionalized CdS colloids] = 3x10-4
mol L-1.
42
0.045
y = 0.001x + 0.005
R2 = 0.9756
0.04
Absorbance
0.035
0.03
0.025
0.02
0.015
0.01
0.005
0
0
10
20
30
40
Concentration (ppm)
Figure 3.4: Standard calibration for functionalized CdS in the presence of various
concentration of BSA. [Functionalized CdS colloids] = 3x10-4 mol L-1.
3.3.2
Fluorescence Properties of the CdS Capped Mercaptoacetic Acid
(Functionalized CdS) Binding with BSA
Bovine serum albumin (BSA) is one of the most available and extensively
studied of all proteins. The study of the interaction fluorescent functionalized CdS
binding with BSA was conducted at room temperature.
The emission spectra of CdS capped with mercaptoacetic acid in the presence
of 25 ppm BSA is shown in Figure 3.5. It was found that the emission maxima of the
fluorescence emission spectra of CdS capped mercaptoacetic acid and BSA is at 350
nm when the excitation peak was at 233 nm wavelengths. It is interesting to note that
the fluorescence properties of functionalized CdS dramatically enhanced when BSA
25 ppm is present in the system. This phenomenon can be attributed to the interaction
between protein (BSA) and functionalized CdS under the room temperature.
This result indicated that this functionalized CdS could be use as a novel
fluorescence probe for the sensitive determination of protein. The functionalized CdS
particles (CdS capped with mercaptoacetic acid) conjugated themselves to protein
43
through covalent interaction. The free carboxylic group from mercaptoacetic acid
was favored for the binding to various biomolecules such as protein by cross-linking
to active amine groups [86].
Functionalized
CdS and BSA
Functionalized
CdS
Figure 3.5: The emission spectra of functionalized CdS in the absence and presence
of BSA under the room temperature. [BSA] = 25 ppm, [Functionalized CdS colloids]
= 3x10-4 mol L-1.
44
3.4
Optimization
Procedures
on
the
Fluorescence
Properties
of
Functionalized CdS Binding with BSA.
3.4.1
Effect of pH Value
Fluorescence variations due to pH changes are caused by different ionizable
chemical species formed by these changes. The changes from these pH variations can
be quite drastic since new ionization forms of the compounds are produced [42].
800
CdS
CdS-BSA
Fluorescence Intensity
700
600
500
400
300
200
100
0
0
2
4
6
pH
8
10
12
Figure 3.6: Effect of pH for functionalized CdS in the absence and presence of BSA
at room temperature. [BSA] = 25 ppm, [Functionalized CdS colloids] = 3x10-4 mol
L-1.
The effect of pH on the fluorescence enhancement of the functionalized CdS
in the absence and presence of BSA was studied (Figure 3.6). Based on result
obtained, it was found that pH does not affect the wavelength of emission spectrum
but effect the fluorescence intensity. The fluorescence emission intensity reached a
maximum over pH 6 and decreased outside of this pH. The pH was only observed in
low pH region for functionalized CdS colloidal solution. In this study, the optimum
pH for the determination of BSA was pH 6.
45
These results seem to indicate that OH- ion is difficult to approach to the CdS
core of the surface modified CdS particles due to the present of the capping agent
(mercaptoacetic acid), which covered the surface of the particles. Uchihara and co
workers [90] supported this conclusion.
Previous studies also have been proposed to explain the appearance of the
result. Uchihara et al. [90] reported that pH dependence was also caused by proton
dissociation equilibrium of the carboxyl group in mercaptoacetic acid. The number
of the mercaptoacetic acid with negative charge increased in the case of CdS capped
with mercaptoacetic acid by increasing the pH. It may cause by mercaptoacetic acid
combined with the CdS surface by the thiol group and the carboxyl group is free for
proton dissociation [90].
3.4.2
Effect of Buffer Nature
A buffer is defined as a mixture of an acid and its conjugate bases, which
may reduce changes in solution pH when acid or alkali, are added. The selection of
an appropriate buffer is important tools in order to maintain a protein at the desired
pH and to ensure reproducible experimental results. Moreover, it is known that
protein structure is very sensitive to environmental factors and easily denatures,
losing its native three-dimensional conformation. This phenomenon occurs due to the
buffers and buffers components can interact directly with certain amino acids and
contribute to the stabilization or destabilization of the compound [22].
Several buffer solutions such as Tris-HCl, NaAc-HCl and BRB-NaOH were
tested in the present experiments. Result obtained was illustrated in Table 3.1. It is
shown that the sensitivity of the protein determination is higher in Tris-HCl (0.1 mol
Lí1) buffer solution than in other buffer meanwhile NaAc-HCl and BRB-NaOH
buffers quenched the fluorescence intensity. In this study, it can be seen that BRB
buffer quenched seriously in the fluorescence intensity. This may be due to the
presence of the phosphate (PO43-) compound in BRB solution, which is a heavy
fluorescence quencher. Therefore, Tris-HCl buffer solution at pH 6 was used in this
46
work. Besides, the pKa value for the Tris buffer is in the range of 8.15, which may
favor the determination of BSA 25 ppm at optimum pH 6.
Table 3.1: The effect of different type of buffer in the presence and absence of BSA.
pH 6, [BSA] = 25 ppm, [Functionalized CdS colloids] = 3x10-4 mol L-1.
Type of Buffer
BRB
NaAc
Tris
3.4.3
Fluorescence Intensity
Absence of Buffer
Presence of Buffer
687.06
570.30
698.87
630.48
698.16
713.80
Effect of Concentration
The concentration of the functionalized CdS colloids may have a great
influence on the fluorescence intensity in the determination of BSA. The effect of the
concentration of the functionalized colloidal solutions has been investigated in
presence of 25 ppm BSA at pH 6. The emission fluorescence intensity increased with
increasing concentration of functionalized CdS (Figure 3.7). The experimental results
indicated that the maximum and constant emission fluorescence intensity occurred
when functionalized CdS colloidal concentration was in the range 2.0x10-4 to 4.0x104
mol L-1. However, the intensity quenched when the concentration of functionalized
CdS colloidal reached 4.5x10-4 mol L-1. Gang et al. [38] suggested that, for a very
dilute solution the relationship between the fluorescence intensity and concentration
is linear. However, at high concentrations the fluorescence intensity decreases
because of the self-quenching. Self-quenching caused by collision of excited
molecules creating radiation less transfer energy [40]. Thus, in this study,
functionalized CdS concentration of 3.0x10-4 mol L-1 was selected for further studies.
47
800
Fluorescence Intensity
700
600
500
400
300
200
100
0
0
1
2
3
4
5
4
Concentration (10 - mol/L)
Figure 3.7: Effect of functionalized CdS concentration on the fluorescence intensity
in the presence of BSA. pH 6, [BSA] = 25 ppm, [Functionalized CdS colloids] =
3x10-4 mol L-1.
3.4.4
Effect of Temperature
All fluorophores are subject to intensity variations as function of temperature.
In general, fluorescence intensity decreases with increasing temperature due to
increasing molecular collision that occurred more frequently at higher temperature.
The degree of response of an individual compound to the temperature variations is
unique to each compound [92].
The response characteristics of the functionalized CdS binding with BSA
were examined at temperatures ranging from 10 oC to 40 oC using BSA 25 ppm test
solutions. Figure 3.8 shows the effect of the reaction temperature on the fluorescence
intensity. It can be seen that the fluorescence intensity is high and constant between
20 oC and 25 oC for functionalized CdS and CdS binding with BSA system. When
the temperature is higher or lower than the optimum range, the relative fluorescence
intensity was decreased. In this study, when the intensity is higher than 30 oC, the
stability of the system decreased and the suitable reaction temperature are in range of
20 oC to 25 oC for the functionalized CdS binding with BSA system. Due to this,
room temperature of 25 oC was selected for further studies.
48
800
CdS+BSA
CdS
Fluorescence Intensity
700
600
500
400
300
200
100
0
0
10
20
30
40
50
0
Temperature ( C)
Figure 3.8: Effect of temperature for functionalized CdS in the absence and
presence of BSA. pH 6, [BSA] = 25 ppm, [Functionalized CdS colloids] = 3x10-4
mol L-1.
3.4.5
Effect of Reaction Time and Stability
Dilute protein solutions often lose activity quickly, possibly because of
denaturation on surface such as glassware. The proteins that denatured may caused
unfolding from their original conformation and apparently from new cross-links
joined the unfolded molecules together in a coagulated mass [31].
In this study, the effect of reaction time and stability of functionalized CdS
binding with BSA was conducted under the room temperature. The time required for
functionalized CdS binding with BSA mixture to bind completely was studied by
measuring the fluorescence intensity at 350 nm for three hours period immediately
after mixing.
Figure 3.9 displays the plot of fluorescence intensity versus effect of reaction
time t (min). Based on the result, it was found that the fluorescence intensity of
functionalized CdS in the absence of BSA was stable but reduced gradually with
49
time and then tends towards a constant value when BSA was present. The
experimental results indicated that the fluorescence intensity quenched rapidly with
increasing reaction time during the first 15 min and the fluorescence intensity
remained constant for at least three hours. This data suggested that the stability of
functionalized CdS binding with BSA system is satisfactory for determination of
BSA. Therefore, the reaction of functionalized CdS binding with BSA was carried
out for 20 min and all the measurements were made within three hours.
1000
Fluorescence Intensity
900
800
700
600
500
400
300
200
100
0
0
50
100
150
200
250
t (min)
Figure 3.9: Effect of reaction time for functionalized CdS in the presence of BSA.
pH 6, [BSA] = 25 ppm, [Functionalized CdS colloids] = 3x10-4 mol L-1.
3.5
Standard Calibration Curves for the Quantitative Analysis of
Functionalized CdS Binding with BSA
Quantitative analysis of proteins is an important task in many fields such as
molecular biology, biotechnology and medical diagnostics. The study of quantitative
analysis of fluorescent functionalized CdS binding with BSA was conducted at room
temperature.
50
In this study, the impacts of various concentration of BSA from 0 ppm to 50
ppm were constructed under the optimum condition such as pH 6 and room
temperature 25 oC. Figure 3.10 shows the intensities of emission spectra of
functionalized CdS at 350 nm wavelengths are significantly enhanced in the
presences of various concentration of BSA. Besides that, a small blue shift was
observed for the emission wavelengths if the concentration of BSA was fixed (Figure
3.10).
The plot of intensity versus concentration (ppm), which is linear, was
obtained as shown in Figure 3.11. A good correlation between BSA concentration
and the fluorescence intensity was obtained with R2 value 0.9899 and the regression
equation was y = 10.56x + 393.89. This result indicated that the functionalized CdS
could be used for the quantitative analysis of BSA (proteins).
Figure 3.10: Emission spectra of functionalized CdS with increasing concentration
of BSA from 0 ppm to 50 ppm. pH 6, [Functionalized CdS colloids] = 3x10-4mol L-1.
51
y = 10.563x + 393.98
R 2 = 0.9899
1000
900
Fluorescence Intensity
800
700
600
500
400
300
200
100
0
0
10
20
30
40
50
60
Concentration (ppm)
Figure 3.11: Calibration curves for functionalized CdS binding with various
concentration of BSA under the optimum condition. pH 6, [Functionalized CdS
colloids] = 3x10-4 mol L-1.
3.6
Method Validation for the Quantitative Analysis of Functionalized CdS
Binding BSA
3.6.1
Linearity of Standard Curves
The calibration graph for functionalized CdS binding with BSA was
constructed from the result obtained under the optimal conditions. In this study, peak
area was linearly proportional to analyte concentration. The calibration graph of
fluorescence intensity versus functionalized CdS with increasing of BSA
concentration expressed in ppm was found to be linear in the range 2 ppm to 50 ppm.
The correlation coefficient of determination is R2 = 0.9899 and regression equation is
y =10.563x + 393.89. It can be concluded that the linearity obtained for the
quantitative analysis of protein was in wider linear range.
52
3.6.2
Accuracy of the Method
Accuracy of the proposed methods was determined in concentration of BSA
25 ppm, by six measurements carried out on different days within three days of
different CdS binding with BSA solutions. For the determination of accuracy of the
developed method, the reproducibility was calculated from 10 independent runs of
25 ppm BSA solution on the same day (Table 3.2). The relative standard deviation
(RSD) obtained for the developed method was 1.5%.
Based on Table 3.3 for inter day accuracy determination, for day 1 the mean
predicted ±SD is 679.49 ± 11.42 with RSD 1.68%, for day 2 the mean predicted ±
SD is 699.79 ± 6.63 with RSD 0.94% and for day 3, the mean predicted ± SD is
703.38 ± 8.95 with RSD 1.27%. Based on the results obtained, it can be concluded
that the method exhibited a high degree of intra and inter-day accuracy.
Table 3.2: Intra-day accuracy of functionalized CdS binding with BSA under
optimum conditions. pH 6, [Functionalized CdS colloids] = 3x10-4 mol L-1.
Replicates
1
2
3
4
5
6
7
8
9
10
Fluorescence Intensity
685.16
690.45
690.53
691.46
672.56
684.32
683.89
675.27
692.10
675.55
Table 3.3: Inter-day accuracy of functionalized CdS binding with BSA under
optimum conditions. pH 6, [Functionalized CdS colloids] = 3x10-4 mol L-1.
Replicates
1
2
3
4
5
6
Day 1
Fluorescence Intensity
Day 2
Day3
690.31
666.94
686.18
692.56
670.03
670.97
691.72
707.79
699.95
704.30
692.02
702.99
702.99
707.62
700.83
702.85
716.72
689.32
53
3.6.3
Limit of the Detection and Quantification
The effect of increasing BSA concentration on the fluorescence intensities of
functionalized CdS was repeated using a lower BSA concentration and at smaller
increment additions to the solution. This study was conducted under the optimal
condition. The increment of the analyte (BSA concentration) is from 1.5 ppm until 4
ppm was carried out. The plot of fluorescence intensity versus concentration of BSA
(ppm) is displayed in Figure 3.12. It can be seen that the fluorescence intensity
enhanced with the increment of the BSA concentration (Figure 3.12). In general,
terms of the limit of detection (LOD) of an analyte can be described as the
concentration that gives an instrument signal (intensity) significantly different from
the blank or background signal. Based on the calibration graph, the limit of detection
for BSA was observed at 1.0 ppm. The 3ı of limit detection measured from blank
measurement for BSA is 0.14 ppm (here ı represents the standard deviation of 10
blank measurements). It can be concluded that this method is suitable for
determining low concentration of BSA and the developed protein assay is a sensitive
assay.
600
Fluorescence Intensity
500
400
300
200
100
0
0
1
2
3
4
5
6
7
Concentration (ppm)
Figure 3.12: Effect of the increasing concentration of BSA on the functionalized
CdS. pH 6, [Functionalized CdS colloids] = 3x10-4 mol L-1.
54
3.7
Salt-Dependent Studies on Functionalized CdS Binding with BSA
Electrostatics is important for BSA binding to the functionalized particles.
The change of the ionic strength is an efficient method for distinguishing the binding
modes between molecules and BSA [69].
In this study, the binding of functionalized CdS with BSA 25 ppm by
influence of salt on the emission intensity at optimum condition was performed. It is
obvious that in the absence of BSA, the addition of NaCl to functionalized CdS
colloidal had an insignificant effect on the fluorescence intensity and in the presences
of BSA, the fluorescence intensity remained constant with increasing of salt
concentration as shown in Figure 3.13.
Based on the previous studies, if the interaction of proteins and reagents was
a result of electrostatic binding, the addition of NaCl reduced the binding of the
reagent to protein and decreased the fluorescence intensity [93]. However, in this
phenomenon, the ionic strength has no effect on this assay. The few effect of NaCl
content on the fluorescence intensity manifested that the interaction of proteins and
reagent was mainly a result of non-electrostatic binding (probably hydrogen bond
bonding) [93]. A hydrogen bonds occurs when two electronegative atoms compete
for the same hydrogen atom. It can be concluded that the binding mechanism of
functionalized CdS and BSA (protein) is non-electrostatic binding. The results
obtained in this study were in good agreement to that reported by other workers [93].
55
1
900
2
Fluorescence Intensity
800
700
600
500
400
300
200
100
0
0
0.1
0.2
0.3
0.4
0.5
0.6
Concentration of NaCl (mol/l)
Figure 3.13: Effect of the concentration of NaCl solution on the fluorescence
intensity in the absence (curve 1) and presence (curve 2) of BSA and functionalized
CdS. [BSA] = 25 ppm, [Functionalized CdS colloids] = 3x10-4 mol L-1.
3.8
Effect of the Foreign Substances on Functionalized CdS Binding with
BSA
In order to test the selectivity of the method for the determination of BSA, a
systematic study of various non-protein substances on the determination of BSA 25
ppm was carried out. Interferences effect may be in the form of suppression or
enhancement of the peak, shifting of the peak, distortion of the peak of interest.
Foreign substances for this determination are amino acids such as L-cysteine, Lalanine, L-tyrosine, L-tryptophan, L-glycine, several metal ions, carbohydrates such
as starch, glucose, sucrose and other compound such as urea and EDTA. The effects
of the respective foreign substances are illustrated in results based on percentage
measurement. Based on results, it can be estimated that the coexisting substances
tested scarcely interfered with the determination of 25 ppm BSA. As a result, the
studies of the interfering substances were carried out at higher concentration.
56
3.8.1
Amino Acids
The graph of fluorescence intensity versus concentration of amino acids
treated with functionalized CdS binding with BSA 25 ppm was plotted as shown in
Figure 3.14. It can be seen that there is no specific trend for the effect of interference
substances on functionalized CdS binding with BSA. Interestingly, the plot showed a
similar trend until the fluorescence intensity of L-cysteine and L-glycine reduced
progressively until the peak was suppressed by 20.8 % and 8.9 %, when the
concentration was at 40 ppm. However, the interference of L-alanine showed an
increment in the fluorescence intensity when the intensity was increased to 80 ppm.
Based on the results obtain, it can be estimated that the presence of L-alanine can be
tolerated at higher concentration but not with L-cysteine and L-glycine. Among the
amino acids, tyrosine and tryptophan have strong native fluorescence and it caused a
high interference on functionalized CdS binding with BSA system.
L-Alanine
L-Glycine
L-Cysteine
900
Fluorescence Intensity
800
700
600
500
400
300
200
100
0
0
20
40
60
80
100
120
Concentration (ppm)
Figure 3.14: Effect of increasing concentration of L-alanine, L-glycine and Lcysteine on functionalized CdS binding with BSA. pH 6, [BSA] = 25 ppm,
[Functionalized CdS colloids] = 3x10-4 mol L-1.
57
3.8.2
Carbohydrates
The presence of starch, glucose, and sucrose were described as in Figure
3.15, graph of fluorescence intensity versus concentration of functionalized CdS
binding with BSA. The fluorescence intensity of glucose and sucrose showed a
similar trend until 30 ppm and decreased when the concentration was 40 ppm. The
peak was suppressed by about 6.0 % and 5.2 % for glucose and sucrose when the
concentration was 10 ppm. However, the interference of starch produced a positive
interference of 8.8 % at 40 ppm concentration. This phenomenon may due to starch a
polymer compound of D-glucose from polysaccharides group. The results showed
that the interference of glucose and sucrose can only be allowed at low concentration
while starch can be allowed at very high concentration.
Starch
Sucrose
Glucose
900
Fluorescence Intensity
800
700
600
500
400
300
200
100
0
0
20
40
60
80
100
120
Concentration (ppm)
Figure 3.15: Effect of increasing concentration of starch, sucrose and glucose on
functionalized CdS binding with BSA. pH 6, [BSA] = 25 ppm, [Functionalized CdS
colloids] = 3x10-4 mol L-1.
58
3.8.3
Metal Ions
Among the ions selected for the interferences study were Ca2+, Cu2+, NH4+,
Mg 2+, Ni 2+, Zn 2+, Fe 2+ and Fe 3+. Several of the ions listed above were tested in the
present study for their ability to reduce or enhance the functionalized CdS binding
with BSA fluorescence peak. The results are given in term of the percentage
measured value in the absence of each metal and are listed in Table 3.4. The data
illustrated that the fluorescence is interfered by the cations. It is assumed that the
decrease in the functionalized CdS binding with BSA fluorescence peak might be
resulted from the interaction of ion with functionalized CdS to form a complex and
the complex interacted with BSA. However, the concentrations of these interferences
ions present in real biological samples are very low [81]. Based on Table 3.4, it can
be seen that the interference of ions such as Ca2+, Ni2+, NH4+, Mg2+, Zn2+, Cu2+ only
can be allowed at very low concentrations. Besides that, as reported by Scope [94]
the presences of divalent cations might accelerated the formation of disulfide bonds.
This phenomenon may cause the ions to reduce the functionalized CdS binding with
BSA fluorescence peak. Further, according to Guibault [40], most of the metal ions
are fluorescence quencher that may cause the fluorescence intensity of the
Fluorescence Intensity
interference ion with functionalized CdS binding with BSA decreased.
800
Ca2+
700
Cu2+
NH4+
600
Ni2+
500
400
300
200
100
0
0
20
40
60
80
100
120
Concentration (ppm)
Figure 3.16: Effect of increasing concentration of Ca2+, Cu2+ NH4+, Ni2+on
functionalized CdS binding with BSA. pH 6, [BSA] = 25 ppm, [Functionalized CdS
colloids] = 3x10-4 mol L-1.
Fluorescence Intensity
59
900
Fe2+
Fe3+
800
Zn2+
Mg2+
700
600
500
400
300
200
100
0
0
20
40
60
80
100
120
Concentration (ppm)
Figure 3.17: Effect of increasing concentration of Fe2+, Fe3+, Zn2+, Mg2+ on
functionalized CdS binding with BSA. pH 6, [BSA] = 25 ppm, [Functionalized CdS
colloids] = 3x10-4 mol L-1.
3.8.4
Other Compounds
Result on the other compounds tested namely EDTA and urea is presented in
Figure 3.18. Based on Figure 3.18, the fluorescence intensity of EDTA and urea were
reduced progressively when the concentration of the both interference substances
was 10 ppm. To illustrate this, the intensity of fluorescence was suppressed about
15.2 % and 5.2 % for EDTA and urea respectively. The result presented shows that
the presence of EDTA and urea only can be allowed at very low concentration.
60
800
EDTA
Urea
Fluorescence Intensity
700
600
500
400
300
200
100
0
0
20
40
60
80
100
120
Concentration (ppm)
Figure 3.18: Effect of increasing concentration of EDTA and urea on functionalized
CdS binding with BSA. pH 6, [BSA] = 25 ppm, [Functionalized CdS colloids] =
3x10-4 mol L-1.
Table 3.4: The effect of foreign substances on the fluorescence intensity of
functionalized CdS binding with BSA 25 ppm.
Substances
Cys
Gly
Ala
Fe (II)
Fe (III)
Ca2+
Cu2+
NH4+
Mg2+
Ni2+
Zn2+
Glucose
Sucrose
Starch
Urea
EDTA
Concentration
(ppm)
50
50
80
50
40
30
10
10
10
30
30
10
10
40
10
10
Enhancement/
Quenching (%)
-20.8
+8.9
+6.5
-4.2
+4.1
-4.8
-16.7
-3.9
+8.1
-8.1
+6.2
-5.2
-6.0
+8.8
-5.2
-15.2
[Percentage of the enhancement and quenching was calculated from the
fluorescence intensity]
61
3.8.5
Surfactant
Detergents are used most often for extraction and purification of membrane
protein, which otherwise are usually insoluble in aqueous solution. Detergents are
amphiphilic molecules with substantial solubility in water. An important property of
detergent is the formation of micelles, which are clusters of detergent molecules in
the hydrophilic head portions face outward. The critical micelle concentration
(CMC) is defined as the lowest detergent concentration at which micelles form. The
classes of detergent can be divided to ionic detergent, nonionic detergent and
zwitterionic detergents [21].
In this studies several surfactant such as ionic detergent sodium dodecyl
sulfate (SDS), nonionic detergent Triton X-100 were selected for the determination
of protein. The interaction with surfactant can be observed by significant changes in
fluorescent intensity or shifts of wavelength in the emission band [95]. Figure 3.19
shows fluorescence emission spectra of functionalized CdS binding with BSA as a
function of SDS concentration. It can be seen that the ionic surfactant SDS decreased
gradually with the fluorescence intensity.
Schweitzer et al. [95] reported similar phenomena as the result obtained
above. It is interesting to note that the addition of SDS to functionalized CdS binding
with BSA solution produced a decrease in the intensity of the fluorescence
concomitant with blue shift of the emission maximum from 350 nm to 330 nm of
wavelength (Figure 3.19). The changes or blue shift in emission band position can be
related to a selective quenching of the more exposed tryptophan group of BSA or
change in the protein conformation [96]. This statement was supported by the study
of Diaz et al. [96] stating that the decreased in the fluorescence intensity of the
functionalized CdS and BSA binding surfactant can be explained in the terms of the
low cooperativity of the surfactant with BSA association. On the other hand, this
phenomenon may be due to the high cooperativity of micelle formation and the
presence of salt would reduce the electrostatic interaction among the surfactant heads
and leaded to the expected decrease in critical micelle concentration (CMC) [96].
Based on the fluorescence emission spectra, graph plot of fluorescence intensity
versus surfactant concentration was obtained as shown in Figure 3.20. The graph
62
illustrated the fluorescence intensity of CdS binding with BSA in presence of SDS
decreased linearly with the concentration.
Figure 3.21 displays the fluorescence emission properties of functionalized
CdS binding with BSA as a function of nonionic surfactant Triton X-100
concentration. In this study, it is interesting to note that the addition of Triton X-100
containing 10 ppm, 20 ppm to the solution CdS binding with BSA 25 ppm produced
an insignificant increment. However, the addition of 30 ppm Triton X-100 showed a
small shift towards shorter wavelength from 350 nm to 315 nm and the intensity
reached a maximum value because of the presence of high concentration 50 ppm of
Triton X-100. This fluorescence intensity increment could be due to an electrostatic
effect and significative cooperativity associated to the formation of micelle like
aggregates on the CdS binding with BSA [97].
Figure 3.19: Fluorescence emission spectra of CdS binding BSA with increasing
concentration of SDS 10 ppm-100 ppm. pH 6, [BSA] = 25 ppm, [Functionalized CdS
colloids] = 3x10-4 mol L-1.
63
1000
SDS
900
Fluorescence Intensity
800
700
600
500
400
300
200
100
0
0
20
40
60
80
100
120
Surfactant Concentaration (ppm)
Figure 3.20: Effect of increasing SDS concentration on the fluorescence emission
intensity of functionalized CdS binding with BSA. pH 6, [BSA]=25 ppm,
[Functionalized CdS colloids] = 3x10-4 mol L-1.
Figure 3.21: Fluorescence emission spectra of functionalized CdS binding with BSA
with increasing concentration of Triton X-100 from 10 ppm to 50 ppm. pH 6, [BSA]
= 25 ppm, [Functionalized CdS colloids] = 3 x10-4 mol L-1.
64
3.9
Standard Calibration of Proteins Binding with Functionalized CdS
Individual responses of various proteins determined in the assay at pH 6 were
carried out. Determination of lysozyme, amylase and egg albumin with
functionalized CdS was carried out and emission spectra are illustrated in Figure
3.22, Figure 3.23 and Figure 3.24 respectively. It can be seen that the emission of
fluorescence in the presences of various proteins remained at the same wavelength
but the intensity are significantly enhanced and the emission bands are symmetric
and narrower than the functionalized CdS.
The calibration graphs for egg albumin, lysozyme and amylase were
constructed under the optimum conditions at room temperature. Based on Figures
3.25, 3.26 and 3.27 the relationship between the fluorescence intensity against the
concentration of several proteins is listed in Table 3.5. A satisfactory linear
relationship with high regression coefficient and a wider linear range are obtained.
As can be seen from Table 3.5, different proteins show different response
characteristics. Long et al. [98] observed similar result. Besides, due the size and the
shape of the protein molecules are different, which may contribute to the differences
in the fluorescence intensity for various proteins [99]. On the other hand, these
phenomena may due to the binding strengths between the functionalized CdS and
different kinds of protein.
Based on the previous studies, it was reported that the intensity of enhanced
fluorescence signals appeared to depend on the electronic properties of the individual
chromophores [38]. Thus, the extent of the electric coupling among chromophores
and the size of the aggregate were formed. It can be seen from Table 3.5 that the
fluorescence sensitivities increased roughly, but not strictly with the increase of isoelectric points (pI) of proteins. This result suggested that electronic coupling plays
the main, but not the only role in the formation of CdS binding with BSA binding
[99]. This result and conclusion were in good agreement with that reported by Gang
et al. [38].
65
Table 3.5: Analytical parameter of various proteins binding with functionalized CdS
under optimum conditions. pH 6, [Functionalized CdS colloids] = 3x10-4 mol L-1.
Proteins
Linear range
Regression equation
pI
r
(ppm)
BSA
2-50
y = 10.56x + 393.89
4.8-4.9
0.9899
Egg albumin
2-30
y = 15.63x + 466.28
4.6-4.7
0.9911
Lysozyme
0.2-12.5
y = 41.86x + 399.91
11.0-11.2
0.9897
Amylase
0.2-7.5
y = 74.60x + 405.53
8.0-9.0
0.9972
Figure 3.22: Emission spectra of functionalized CdS with increasing concentration
of lysozyme from 0 ppm to 12.5 ppm. pH 6, [Functionalized CdS colloids] = 3x10-4
mol L-1.
66
Figure 3.23: Emission spectra of functionalized CdS with increasing concentration
of amylase from 0 ppm to 10 ppm. pH 6, [Functionalized CdS colloids] = 3x10-4 mol
L-1.
Figure 3.24: Emission spectra of functionalized CdS with increasing concentration
of egg albumin from 0 ppm to 30 ppm. pH 6, [Functionalized CdS colloids] = 3x10-4
mol L-1.
67
y = 41.857x + 399.91
1000
R2 = 0.9897
Fluorescence Intensity
900
800
700
600
500
400
300
200
100
0
0
2
4
6
8
10
12
14
Concentration (ppm)
Figure 3.25: Calibration graph for functionalized CdS binding with protein lysozyme
under the optimum conditions. pH 6, [Functionalized CdS colloids] = 3x10-4 mol L-1.
y = 74.6x + 405.53
R2 = 0.9972
Fluorescence Intensity
1200
1000
800
600
400
200
0
0
2
4
6
8
Concentration (ppm)
Figure 3.26: Calibration graph for functionalized CdS binding with protein amylase
under the optimum conditions. pH 6, [Functionalized CdS colloids] = 3x10-4 mol L-1.
Fluorescence Intensity
68
y = 15.631x + 446.28
R2 = 0.9911
1000
900
800
700
600
500
400
300
200
100
0
0
5
10
15
20
25
30
35
Concentration (ppm)
Figure 3.27: Calibration graph for functionalized CdS binding with egg albumin
under the optimum conditions. pH 6, [Functionalized CdS colloids] = 3x10-4 mol L-1.
3.10
Comparison of the Developed Functionalized CdS Methods
The characteristic of the developed method functionalized CdS binding with
BSA and other established method for the protein determination are illustrated in
Table 3.6. Perez-Ruiz et al. [53] developed the lower fluorescence of Rose Bengal
(RB) caused by binding of the dye to the proteins. In this study, the decreased in the
fluorescence intensity, measured at emission 572 nm wavelength when excitation at
555 nm wavelength, was linearly related to protein concentration from 1.3 to 24.5
ppm. The detection limit was 0.3 ppm. It is widely accepted that the (RB) method is
sensitive method due to low limit of detection value in BSA determination. However,
this method seems to have smaller Stoke’s shift (17 nm) compared to developed
functionalized CdS method with higher value of Stoke’s shift (117 nm).
Chun et al. [50] described fluorescence quenching of Erythrosin B (EB) by
proteins method. This method resulted with a very low limit of detection, which may
favor to the sensitivity of the method. However, the novel developed method CdS
have a wider linear range (0.2-50 ppm) for the protein determination compared to the
EB method. The interactions between terazosin and BSA method were developed by
69
fluorescence study was proposed by Jiang et al. [46]. The study of the quantitative
analysis of the method resulted high LOD value of 0.21 ppm compared to the novel
developed CdS method, which was sensitive method with low LOD value of 0.14.
In brief, the developed method showed a higher sensitivity and a wider linear
range and good precision compared to organic dyes binding method. In addition, the
organic dyes are expensive compared to inorganic compound CdS. The proposed
CdS binding with BSA method have a larger Stoke’s value.
Table 3.6: Comparison of methods for the determination of protein (BSA).
Method
Ȝem/Ȝex (nm)
LOD (ppm)
Linear range (ppm)
RB
572/555
0.3
1.3-24.5
EB
550/317
0.06
1.36-20.40
Terazosin
280/413
0.21
This method
233/350
0.14
3.11
2-50
R.S.D (%)
0.2-0.79
2.5
1.5
Conclusions
The fluorescence method based on functionalized CdS binding with BSA
(protein) through covalent coupling with carboxyl group has been successfully
developed. Under the optimum conditions, the calibration graph for the BSA
determination is a linear graph with R2 value 0.9899. In addition to its sensitivity, the
limit of detection was 0.14 ppm. As conclusion, the developed protein assay is highly
reproducible, relatively large Stoke’s shift, wider linear range and good precision.
CHAPTER 4
FUNCTIONALIZED ZINC SULFIDE BINDING BSA
4.1
Preamble
ZnS capped L-cysteine (functionalized ZnS) particles was produced by colloidal
aqueous synthesis, in which sulfide was introduced into zinc-cysteine solution. In
this study, the synthesis method produced a white colloidal ZnS capped L-cysteine.
L-cysteine, which is a water-soluble sulfhydryl-containing amino acid, was used as
stabilizer compound for the ZnS particles. In this chapter, the interaction between
ZnS capped cysteine (functionalized ZnS) and BSA was established by fluorescence
method. The optimization procedure was carried for the assay of BSA. The
qualitative and quantitative analysis of BSA by using fluorescence method was
discussed.
71
4.2
Fundamental Studies of ZnS Capped L-Cysteine (Functionalized ZnS)
4.2.1
Spectral Characteristics of Fluorescence on Functionalized ZnS
Fluorescence
spectrophotometer
studies
on
ZnS
capped L-cysteine
(functionalized ZnS) has been conducted in order to obtain two spectra, namely
emission spectra and excitation spectra. The fluorescence emission spectra of
functionalized ZnS were recorded from 200 nm to 800 nm wavelength using
luminescence spectrometer LS 50B at room temperature. The result given in
Figure 4.1 shows that the emission spectra of functionalized ZnS at 357 nm
wavelength when the excitation peak was at 233 nm. The emission spectrum of
functionalized ZnS obtained was broad band emissions. According to Sander et al.
[63], the large spectral width emission band was caused by both inhomogeneous
broadening (due to a variation in particles size) and broadening due to electron
phonon coupling.
The slit width used for this fluorescence measurement was 5 nm. The slit
width could not be increase very much, because the slit width of more than 5 nm
caused the detector signal to be over-range, resulted in arbitrary fluorescence
intensity above 1000 nm. In this study, the large interval (> 150 nm) between the
excitation wavelength (233 nm) and the emission wavelength (357 nm) is beneficial
to the sensitivity and accuracy of this method because fluorescence from the
background and the scattering light can be greatly reduced. This statement made was
in good agreement with those obtained by Li et al. [47].
72
Figure 4.1: Fluorescence excitation spectrum(a) and emission spectrum(b) for ZnS
capped cysteine. (Ȝexc = 233 nm, [Functionalized ZnS colloids] = 4x10-4 mol L-1).
4.3
Standard Calibration of Absorbance Properties on Functionalized
ZnS Binding with BSA
The UV absorbance spectra of ZnS capped L-cysteine (functionalized ZnS)
and BSA was obtained under the room temperature using the Shimadzu UV-visble
spectrophotometer-1601PC. The UV spectra of functionalized ZnS in the presence of
various concentration of BSA are shown in Figure 4.2. In this study, Ȝmax around 280
nm was used to quantify the interaction of functionalized ZnS in presence of BSA.
Based on Figure 4.2, it is interesting to note that the absorbance of functionalized
ZnS is about 0.012 in the absence of BSA. This absorbance occurred may be due to
the presence of capping material L-cysteine compound of amino acid group. As seen
in Figure 4.2, the absorbance of functionalized ZnS increased with increasing amount
of concentration of BSA from 5 ppm to 35 ppm. The absorption at the near UV range
280 nm wavelength was obtained is caused by the amino acid residues that presence
in the proteins (BSA).
73
Based on the absorbance spectra obtained from the series of different
concentration of BSA (Figure 4.3), a plot of absorbance versus concentration was
obtained. A good correlation between BSA concentration and the UV absorbance
was observed and the linear curve agrees with Beer’s law with R2 value 0.9755 and
regression equation with y = 0.0009x + 0.0105 as shown in Figure 4.3.
Figure 4.2: UV absorbance spectra of functionalized ZnS binding with BSA
concentration 0 ppm to 35 ppm. ([Functionalized ZnS colloids] = 4x10-4 mol L-1).
y = 0.0009x + 0.0105
R2 = 0.9755
0.045
0.04
Absorbance
0.035
0.03
0.025
0.02
0.015
0.01
0.005
0
0
10
20
30
40
Concentration (ppm)
Figure 4.3: Standard calibration for functionalized ZnS in presence of various
concentration of BSA.( [Functionalized ZnS colloids] = 4x10-4 mol L-1).
74
4.4
Fluorescence Properties of ZnS Capped L–Cysteine (Functionalized
ZnS) binding with BSA
Figure 4.4: The emission spectra of functionalized ZnS in the absence and presence
of BSA under the room temperature. [BSA] = 15 ppm, [Functionalized ZnS colloids]
= 4x10-4 mol L-1.
The fluorescence properties of the interaction between ZnS capped L-cysteine
(functionalized ZnS) and BSA was conducted under the room temperature. Figure
4.4 shows the behavior of emission spectra of functionalized ZnS binding with BSA.
The fluorescence properties of emission wavelength were at 357 nm wavelengths
when the excitation peak was at 233 nm wavelength as shown in Figure 4.4. It is
interesting to note that the impact of 15 ppm concentration of BSA on the
functionalized ZnS system is obviously different. In this study, the fluorescence
intensity of functionalized ZnS was significantly enhanced from fluorescence
intensity of 394.00 to 720.37 in the presence of BSA 15 ppm. However, the
wavelength of the emission spectra functionalized ZnS remained at 357 nm in the
presence of 15 ppm BSA. The emission spectra above indicated that BSA has the
75
best enhancing effect on functionalized ZnS. Therefore, the enhancement of emission
intensity of the mixture of functionalized ZnS and BSA suggested that interaction of
functionalized ZnS and BSA had taken place.
Previous studies have been proposed to explain these phenomena. According
to Kho et al. [78], it can be deduced that the L-cysteine has been covalently linked to
the surface of ZnS and formed the L-cysteine capped ZnS particles [78] as shown in
Figure 4.5. Further L-cysteine binds to a Zn atom, the polar carboxyl group is
available for covalent coupling to various biomolecules such as protein and nucleic
CH 2 CH(NH 2 )COOH
acids by cross-linking to active amine group (Figure 4.6) [81].
s
s
ZnS
s
CH2CH(NH2)COOH
CH2CH(NH2)COOH
s
CH 2 CH(NH 2)COOH
CH2CH(NH2)COOH
Figure 4.5: Schematic of ZnS capped L-cysteine
s
O
s
ZnS
s
CH2CH(NH2)CN
Protein
CH2CH(NH2)COOH
H
s
CH2CH(NH 2)COOH
Figure 4.6: Schematic of functionalized ZnS conjugated to protein
76
4.5
Optimizations
Procedures
on
the
Fluorescence
Properties
of
Functionalized ZnS Binding with BSA
Chemical variables such as effect of pH, concentration, temperature and
reaction time were performed and optimized to obtain the optimum, best
measurement conditions and stable fluorescent signal.
4.5.1
Effect of pH Value and Buffer Nature
The influence of acidity and alkalinity of the medium on the intensity of
fluorescence were studied for each compound. The effect of the pH on the
fluorescence intensity of functionalized ZnS in the absence and presence of BSA was
studied in the pH range of 2.0 to 11.0. In an attempt to see the relationship between
fluorescence intensity and pH, the fluorescence intensity was plotted as a function of
pH value (Figure 4.7). It was observed that in the lower pH range pH 2 to 5 the
fluorescence intensity increases with an increase of pH. In the higher value range of
pH 9-11, the fluorescence intensity decreases linearly. Hence, it is widely accepted
that the change of pH values affect the sulfhydryl-containing amino acid, cysteine
coordinating to Zn2+ ions, which cause the different pH growth process of ZnS
capped cysteine [100]. Besides, in the presence of BSA, the pH system occurred at
maximum fluorescence intensity in the range of pH 6 to pH 8. In this study, the
optimum pH of functionalized ZnS binding with BSA determination was selected at
pH 7.
From pH 9 to pH 11, the intensity is decreasing gradually due to the created
of OH- ions. These result may be due to OH- ion is difficult to approach to the
surface of functionalized ZnS particles because of the presence of the capping agent
(cysteine) which cover the surface of the particles [90].
These results obtained do not contradict with that obtained by Hao et al.
[100] when they studied the effect of the pH of ZnS capped mercaptoacetic acid. In
their studies, the pH sharply increased at lower pH range 2.0 to 5.0. The result shows
77
that the fluorescence intensity of functionalized ZnS is totally depends on the pH of
the capping material, which involves the reaction of ZnS capped mercapto acetic
acid.
ZnS
800
ZnS-BSA
Fluorescence Intensity
700
600
500
400
300
200
100
0
0
2
4
6
8
10
12
pH
Figure 4.7: Effect of pH for functionalized ZnS in absence and presence of
BSA at room temperature. [BSA] = 15 ppm, [Functionalized ZnS colloids] =
4x10-4 mol L-1.
Labeling protein should be carried out in a well-buffered system at a pH that
is optimal for the reaction. Additionally, the effect of buffer nature on the
fluorescence intensity was examined. In this study, several buffer solutions (Tris–
HCl, NaAc–HCl, BRB–NaOH) were selected. The result illustrated in Table 4.1
indicates that the sensitivity of the protein determination is higher in Tris–HCl (0.1
mol Lí1) buffer solution compared to NaAc and BRB buffer. However, NaAc and
BRB buffer was quenched in the protein determination. This may be due to the
formation of insoluble complexes and chelation of the metal with functionalized ZnS
and BSA. The acetate buffer has a very low pKa value 4.76, which might not be
favorable in the protein determination at optimum pH 7. Moreover, according to
Grady et al. [101], phosphate buffer is a feeble buffer in pH range 8-11. Therefore,
Tris–HCl buffer solution at pH 7 was recommended for further study.
78
Table 4.1: The effect of different type of buffer in the presence and absence of BSA.
pH 7, [BSA] = 15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1.
Type of Buffer
BRB
NaAc
Tris
4.5.2
Fluorescence Intensity
Absence of Buffer
Presence of Buffer
692.82
650.94
676.06
651.44
667.68
684.69
Effect of Concentration
Fluorescence are linearly related to concentration at low concentration
(10-9 to 10-6). However, at high concentrations the fluorescence intensity decreases
because of self–quenching and due to inner cell effect [40].
The emission fluorescence intensity was measured over the concentration of
functionalized ZnS in the range of 1 x 10-4 mol L-1 to 8 x 10-4 mol L-1 to investigate
the effect of concentration for BSA determination. The study was conducted at pH 7
and at room temperature. Based on the result obtained, graph fluorescence intensity
versus concentration of functionalized ZnS in presence of BSA was plotted (Figure
4.8). The graph shows that the fluorescence intensity of functionalized ZnS increases
linearly with the concentration and reaches the optimum range at 4 x 10-4 to 6 x 10-4
mol L-1 in the presence of BSA. However, further increase in the concentration of
functionalized ZnS caused the fluorescence intensity to decrease. This may be due to
functionalized ZnS tends to precipitate when the concentration of colloidal was more
than 7 x 10-4 mol L-1. Based on result obtained it was found that the concentration of
functionalized ZnS colloidal 4 x 10-4 mol L-1 was selected as optimum concentration
for the BSA determination.
79
800
Fluorescence Intensity
700
600
500
400
300
200
100
0
0
2
4
6
8
10
4
Concentration (10 - mol/L )
Figure 4.8: Effect of functionalized ZnS concentration on the fluorescence intensity
in the presence of BSA. pH 7, [BSA] = 15 ppm, [Functionalized ZnS colloids] =
4x10-4 mol L-1.
4.5.3
Effect of Temperature and Reaction Time
Optimization of the temperature effect is one of the important tools in protein
analysis. The binding properties between functionalized ZnS with BSA were
determined at different temperatures (10, 15, 20, 25, 30, 35, 40 oC). Based on the
results, a plot of fluorescence intensity versus temperature is illustrated in Figure 4.9.
The data appeared that the increasing temperature effect showed a minimal effect on
the fluorescence intensity. It was illustrated that the temperature of functionalized
binding BSA was greatest at 25 oC as shown in Figure 4.9. However, the
fluorescence intensity decreases sharply with increasing temperature from 30 oC until
40 oC. Due to this, room temperature 25 oC was adopted for further study.
In this studies the changes in the fluorescence intensity is in range of 2 % to 8
% per 5 oC was obtained. The result of this study was in good agreement with an
earlier report, which suggested that the change in fluorescence intensity could be as
high as 5 % per 5 oC [40]. However, the result obtained did contradict with previous
80
statement, which stated that as the temperature is increased, the fluorescence
intensity decreases [40].
The effect of reaction time on functionalized ZnS binding with BSA was
conducted under the room temperature. Based on the graph obtained fluorescence
intensity versus reaction time (min) (Figure 4.10), it was found that the binding
between functionalized ZnS with BSA proceeds rapidly at room temperature and the
intensity reached the constant value within 20 min and remained constant for 5 hours
(Figure 4.10). Due to this, the reaction of functionalized ZnS binding with BSA was
carried out for 20 min and all the measurement were made within 5 hours for further
studies.
Based on the result obtained, it is generally agreed that the stability of
functionalized ZnS binding with BSA are more stable because the intensity remains
constant for 5 hours. It is widely accepted that ZnS capped with cysteine are more
stable compared to ZnS capped with mercaptoacetic acid. This conclusion agrees
with the statement reported by Li et al. [81] that comparison with using
mercaptoacetic acid, as stabilizer cysteine, is more stable.
ZnS
700
ZnS+BSA
Fluorescence Intensity
600
500
400
300
200
100
0
0
10
20
30
40
50
o
Temperature ( C)
Figure 4.9: Effect of temperature for functionalized ZnS in absence and presence of
BSA. pH 7, [BSA] = 15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1.
81
900
Fluorescence Intensity
800
700
600
500
400
300
200
100
0
0
100
200
300
400
500
min (t)
Figure 4.10: Effect of reaction time for functionalized ZnS in the presence of BSA.
pH 7, [BSA] = 15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1.
4.6
Standard Calibration Curves for the Quantitative Analysis of
Functionalized ZnS Binding with BSA
The study of the binding system between the functionalized ZnS and BSA
was conducted under room temperature. Under the optimum condition, a study was
made of the relationship between emission spectra and various concentration of BSA
for the quantitative analysis of BSA. Figure 4.11 shows the emission spectra of
functionalized ZnS with various concentration of BSA.
Based on the result, it was found out that the emission maxima of the
fluorescence spectra of functionalized ZnS binding with BSA system are similar to
that of functionalized ZnS but the intensities are significantly enhanced (Figure
4.11). Thus, a small blue shift of the emission spectra was observed from the
wavelength of functionalized ZnS at 357 nm to 350 nm for functionalized ZnS
binding with BSA. This is may be due to energy transfer of the system [102].
Further, this results indicated that the lower the dielectric constants of the solvent
water, the shorter is the wavelength of emission, suggesting a much reduced dipole-
82
dipole interaction between the fluorescent molecules and the BSA solvent occurred
[103]. From Figure 4.11, it can be observed that the peak intensiy tend to be leveled
off with addition of BSA may be due to over range of fluorescence intensity.
Figure 4.11: Emission spectra of functionalized ZnS with increasing concentration of
BSA from 2.5 ppm to 20 ppm. pH 7, [Functionalized ZnS colloids] = 4x10-4 mol L-1.
The impact of various concentrations 0.2 ppm to 22.5 ppm of BSA on the
functionalized ZnS system is obviously different. In brief, the data indicated that
BSA has the best enhancing effect on functionalized ZnS system for the quantitative
analysis of proteins. Based on data obtained, the calibration curve for BSA was
constructed from the result obtained under the optimal condition. Figure 4.12 shows
the plot of intensity versus concentration (ppm) is linear graph with (R2 = 0.9805, n =
12). It also shows that the range of linearity was found to be from 0.2 ppm to 22.5
ppm of BSA.
83
1200
y = 21.886x + 456.05
R2 = 0.9805
Fluorescence Intensity
1000
800
600
400
200
0
0
5
10
15
20
25
Concentration (ppm)
Figure 4.12: Calibration curves for functionalized ZnS binding with various
concentration of BSA under optimum conditions. pH 7, [Functionalized ZnS
colloids] = 4x10-4 mol L-1.
4.7
Method Validation for the Quantitative Analysis of BSA
4.7.1
Linearity, Accuracy, Limit of the Detection of the Method
The calibration graph of fluorescence intensity functionalized ZnS with
increasing of BSA concentration expressed in ppm was found to be linear in the
range of 0.4 to 22.5 ppm with R2 = 0.9805 and linear equation is y = 21.866x +
456.05. The reproducibility of the developed method calculated from six independent
runs of 15 ppm of the BSA produced the relative standard deviation (RSD) of 1.7%.
For inter-days accuracy determination, for day 1 the mean predicted ±SD is 733.52 ±
4.923 with RSD 0.67%, for day 2 the mean predicted ±SD is 707.08 ± 9.19 with
RSD 1.30% and for day 3 the mean predicted ± SD is 695.19 ± 8.48 with RSD
1.21% respectively. Here, the peak areas obtained were reproducible, as the
maximum RSD observed was 1.7%.
84
Limit of detection is one of major part in quantitative analysis of the
developed method to obtain the minimum concentration of analyte for the
determination. In order to identify the sensitivity of the develop method, the study of
limit of detection was carried out under the optimum conditions. In this study, the
effect of BSA concentration on functionalized ZnS fluorescence intensity was
repeated using lower concentration of BSA and small increment from 0.2 ppm until
4 ppm. The calibration graph of fluorescence intensity versus concentration (ppm) of
BSA was obtained as shown in Figure 4.13. It can be seen that the fluorescence
intensity is directly proportional to the concentration of protein and the signal of the
fluorescence intensity are significantly different from the background signal at 0.2
ppm BSA concentration. However, the 3ı of limit detection measured from blank
measurement for BSA was 0.09 ppm. Therefore, it can be concluded that, this
method has high sensitivity, high accuracy and a wider linear range.
700
Fluorescence Intensity
600
500
400
300
200
100
0
0
1
2
3
4
5
6
7
Concentration (ppm)
Figure 4.13: Effect of the increasing concentration of BSA on the functionalized
ZnS. pH 7, [Functionalized ZnS colloids] = 4x10-4 mol L-1.
85
4.8
Salt Dependence Studies on Functionalized ZnS Binding with BSA
Small molecules are bound to macromolecule by four binding modes such as
H-bond, van der Waals, electrostatic and hydrophobic interactions. The study of the
effect of salt dependence of functionalized ZnS binding with BSA on fluorescence
intensity of emission has led to some interesting results. As shown in Figure 4.14 it is
obvious that in the absence of BSA, the addition of NaCl to colloidal has no
significant effect on the fluorescence intensity. However, in the presence of BSA the
fluorescence intensity increases with increasing of salt concentration (Figure 4.14).
Previous studies reported that if the interaction of proteins and reagents were
a result of electrostatic binding, the addition of NaCl would reduce the binding of the
reagent to protein and result in a decreased the fluorescence intensity [34]. However,
in this study the increment of the NaCl concentration favors in increasing the
fluorescence intensity. Here, it is generally agreed that the binding mode between
functionalized ZnS with BSA mainly results of non-electrostatic binding. This result
indicated that the binding modes between the reagent and BSA might be due to
hydrophobic binding because of the presences of the nonpolar group ZnS capped
cysteine.
1
1000
2
900
Fluorescence Intensity
800
700
600
500
400
300
200
100
0
0
0.1
0.2
0.3
0.4
0.5
0.6
Concentration of NaCl (mol/L)
Figure 4.14: Effect of the concentration of NaCl solution on the fluorescence
intensity in the absence (curve 1) and presence (curve 2) of BSA and functionalized
colloidal ZnS. [BSA] = 15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1.
86
4.9
Effect of the Foreign Substances on Functionalized ZnS Binding with
BSA
4.9.1
Effect of Non-protein Substances
Human body fluids contain various non-protein substances such as amino
acid such as L-cysteine (Cys), L-alanine (Ala), L-tyrosine (Tyr), L-glycine (Gly), Ltryptophan (Trp), glucose, sucrose, starch and other compound such urea, metal ions
and EDTA. In this study, the effect of several non-protein compounds and metals are
successfully treated with functionalized ZnS binding with BSA 15 ppm to reduce
their concentration below the maximum allowable limits for the respective nonprotein substances.
The results are given in terms of the percentage-measured value in the
presence of each non-protein compound in the Table 4.2. Various metal ions and
non-protein substance have different influences on the functionalized ZnS binding
with BSA. Graphs of fluorescence intensity versus concentration of metal ions and
non-protein substance were plotted as shown in Figures 4.15 to 4.19 respectively.
Based on Figure 4.15, the interference of L-cysteine, L-glycine and L-alanine
seems to be very high because the intensity decreases more gradually and highly
interfered in a very low concentration. Besides, L-tyrosine and L-tryptophan showed a
maximum interference on functionalized ZnS binding with BSA due to the strong
native fluorescence of the compound. Thus, these compounds only can be allowed at
very low concentration. The result obtained indicates that formation between amino
acid compounds and ZnS capped L-cysteine may occur. Besides, glucose, sucrose
and starch can be allowed at higher concentration because of minimal effect on
functionalized ZnS binding with BSA system (Figure 4.16). Table 4.2 illustrates that
the glucose, sucrose and starch only interfered in the range of 3.5 % to 7.0 % when
the concentration was increased up to 50 ppm.
On the other hand, the presence of various metals, urea and EDTA was
illustrated in Figures 4.17, 4.18 and 4.19. Based on Figure 4.18, it is interesting to
note that the Fe3+ ion eliminates the functionalized ZnS with BSA emission peak
87
even when it is present at relatively low concentration. This is may be due to the
Fe3+ ion formed a colloid or precipitated with functionalized ZnS and BSA under the
experimental conditions. Fe2+ ion and Cd2+ ion interfered at about 7.0 to 8.6%, if the
concentration is above 10 ppm. In the case of Cu2+ ion the emission peak decreased
about 26.5% in presence of low concentration 10 ppm of Cu2+ ion followed by Fe3+
ion interfered about 66.6% when the concentration is 10 ppm. The other seven metal
ions tested, namely Cu2+, NH4+, Mg2+, Ni2+, Ca2+ only have a slight interference on
functionalized ZnS binding with BSA. Based on Table 4.2, NH4+, Mg2+, Ni2+and
Ca2+ ions, EDTA and urea did not interfere when present at low concentration but the
emission intensity was suppressed slightly by about 7.0 to 9.4% when ion
concentration was increased to 40 ppm. In this study, it was found that Cu2+ ion and
Fe3+ ion only could be allowed at relatively low concentration. However, the
concentration of these interferences ions is very low in real biological samples [8].
Table 4.2: Effect of foreign substances on the fluorescence intensity of
functionalized ZnS binding with BSA 15 ppm.
Substances
Concentration (ppm)
Cys
Gly
Ala
Fe (II)
Fe (III)
Ca2+
Cu2+
NH4+
Mg2+
Ni2+
Cd2+
Glucose
Sucrose
Starch
Urea
EDTA
10
30
20
10
10
30
10
30
30
40
10
50
50
50
40
30
Quenching/Enhancement (%)
-8.9
-9.1
-7.7
-8.6
-66.6
-7.8
-26.5
-8.2
-7.6
-7.0
-7.4
-7.0
-3.7
-5.8
-9.8
-6.4
[Percentage of the enhancement and quenching was calculated from the
fluorescence intensity]
88
L-Glycine
800
L-Cysteine
L-Alanine
Fluorescence Intensity
700
600
500
400
300
200
100
0
0
20
40
60
80
100
12 0
Concentration (ppm)
Figure 4.15: Effect of increasing concentration of L-alanine, L-glycine and Lcysteine on functionalized ZnS binding with BSA. pH 7, [BSA]=15 ppm,
[Functionalized ZnS colloids] = 4x10-4 mol L-1.
Glucose
800
Sucrose
Fluorescence Intensity
700
Starch
600
500
400
300
200
100
0
0
20
40
60
80
100
120
Concentration (ppm)
Figure 4.16: Effect of increasing concentration of starch, sucrose, glucose on
functionalized ZnS binding with BSA. pH 7, [BSA] = 15 ppm, [Functionalized ZnS
colloids] = 4x10-4 mol L-1.
89
Cu2+
NH4+
Ni2+
Mg2+
800
Fluorescence Intensity
700
600
500
400
300
200
100
0
0
20
40
60
80
100
120
Concentration (ppm)
Figure 4.17: Effect of increasing concentration of Mg2+, Cu2+ NH4+and Ni2+ on
functionalized ZnS binding with BSA. pH 7, [BSA] = 15 ppm, [Functionalized ZnS
colloids] = 4x10-4 mol L-1.
Cd2+
800
Fe2+
Fluorescence Intensity
700
Fe3+
Ca2+
600
500
400
300
200
100
0
0
20
40
60
80
100
120
Concentration (ppm)
Figure 4.18: Effect of increasing concentration of Fe2+, Fe3+ Cd2+and Ca2+ on
functionalized ZnS binding with BSA. pH 7, [BSA] = 15 ppm, [Functionalized ZnS
colloids] = 4x 10-4 mol L-1.
90
EDTA
800
Urea
Fluorescence Intensity
700
600
500
400
300
200
100
0
0
20
40
60
80
100
120
Concentration (ppm)
Figure 4.19: Effect of increasing concentration of EDTA and urea on functionalized
ZnS binding with BSA. pH 7, [BSA] = 15 ppm, [Functionalized ZnS colloids] =
4 x10-4 mol L-1.
4.9.2 The Effect of Surfactant
4.9.2.1 Sodium Dodecyl Sulfate (SDS)
The effect of surfactants, sodium dodecyl sulfate (SDS), nonionic detergent
Triton X-100 on functionalized ZnS binding with BSA emission peak was studied.
Aliquots of SDS were added to solution of functionalized ZnS binding with BSA 15
ppm. The result obtained (Figure 4.20) shows that SDS did not interfere with the
determination from up to a concentration of 20 ppm. In the presence of higher
concentration of SDS the wavelength of maximum emission was modified. In
particular, small shift towards shorter wavelengths in the presence of SDS was
observed. The position of the centre of the fluorescence emission band changed from
350 nm to 340 nm wavelengths when the surfactant concentration was 30 ppm
(Figure 4.20).
91
On the other hand, further addition of SDS, caused the emission intensity
decreased progressively. This type of phenomena may be due to an over folding of
the protein resulting from the surfactant association [104]. Presence of high
concentration of oppositely charged an anionic surfactant SDS plays the role of
supplying negative charges to neutral charge of protein. This phenomenon may
prevent the functionalized ZnS binding with protein (BSA) and the emission
intensity of the reaction system decrease [105].
Figure 4.20: Fluorescence emission spectra of ZnS binding BSA with increasing
concentration of SDS 10 ppm-100 ppm. pH 7, [BSA]=15 ppm, [Functionalized ZnS
colloids] = 4x10-4 mol L-1.
4.9.2.2 Triton X-100
Similar experiment as performed for the SDS surfactant, Triton X-100 was
used as a non-ionic surfactant for the functionalized ZnS binding with BSA. A more
significant variation was encountered in Triton X-100 when the surfactant was added
in functionalized ZnS binding with BSA 15 ppm. Based on Figure 4.21 a significant
92
shift towards a shorter wavelength from 350 nm to 311 nm wavelengths was
observed. The intensity value caused arbitrary fluorescence intensity above 1000
when the concentration of surfactant was 30 ppm. The changes in emission
wavelength can be related to changes in the protein conformation and from surfactant
association. This conclusion was in good agreement with those obtained by
Moriyama et al. [106].
Figure 4.21: Fluorescence emission spectra of functionalized ZnS binding with BSA
with increasing concentration of Triton X-100 from 10 ppm to 30 ppm. pH 7, [BSA]
= 15 ppm, [Functionalized ZnS colloids] = 4x10-4 mol L-1.
4.10
Standard Calibration of Proteins Binding with Functionalized ZnS
Fluorescence studies were carried out to observe the behaviour between
functionalized ZnS with different type of proteins such as BSA, egg albumin,
lysozyme and amylase under the optimum conditions. The emission spectra of
various proteins amylase, lysozyme and egg albumin are shown in Figures 4.22, 4.23
and 4.24 respectively. The result shows a similar fluorescence emission peak were
observed at 357 nm wavelengths for the protein amylase, lysozyme and egg albumin.
93
However, the result obtained shows a difference in the response of the different type
of protein. Based on Figures 4.25, 4.26 and 4.27 it can be observed that the
fluorescence intensity is directly proportional to the concentration of the various
proteins in a certain range depending on different protein system.
Table 4.3 summarized the results obtained from the experiments carried out
on the various compound of proteins. A satisfactory linear relationship with high
regression coefficient and a wider linear range are obtained. Table 4.3 summarized
the results obtained from the experiments carried out on the various compound of
proteins. As shown in Table 4.3, the linear range for egg albumin, lysozyme and
amylase are 0.2-20 ppm, 0.2-10 ppm, 0.2-20 ppm and 0.2-7.5 ppm respectively and
the coefficients of correlation are above 0.9367.
The result indicated that various possibilities: either different protein has
different isoelectric points or the weight, size and shape of the molecules are also
different, so the fluorescence intensity for various proteins is different [107].
Table 4.3: Analytical parameter of various proteins binding with functionalized ZnS
under the optimum conditions. pH 7, [Functionalized ZnS colloids] = 4x10-4 mol L-1.
Proteins
Linear
Regression equation
pI
R2
range (ppm)
BSA
0.4-22.5
y = 21.886x+456.05
4.8-4.9
0.9805
Egg albumin
0.2-20
y = 21.911x+479.13
4.6-4.7
0.9367
Lysozyme
0.2-10
y = 45.04x+501.21
11.0-11.2
0.9851
Amylase
0.2-7.5
y = 51.04x+495.42
8.0-9.0
0.9877
94
Figure 4.22: Emission spectra of functionalized ZnS with increasing concentration
of amylase from 0 ppm to 7.5 ppm. pH 7, [Functionalized ZnS colloids] = 4x10-4 mol
L-1.
Figure 4.23: Emission spectra of functionalized ZnS with increasing concentration
of lysozyme from 0 ppm to 10 ppm. pH 7, [Functionalized ZnS colloids] = 4x10-4
mol L-1.
95
Figure 4.24: Emission spectra of functionalized ZnS with increasing concentration
of egg albumin from 0 ppm to 20 ppm. pH 7, [Functionalized ZnS colloids] = 4x10-4
mol L-1.
y = 51.047x + 495.42
1000
R2 = 0.9877
900
Fluorescence Intensity
800
700
600
500
400
300
200
100
0
0
1
2
3
4
5
6
7
8
Concentration (ppm)
Figure 4.25: Calibration graph for functionalized ZnS binding with protein amylase
under optimum conditions. pH 7, [Functionalized ZnS colloids] = 4x10-4 mol L-1.
96
y = 45.043x + 501.21
R 2 = 0.9851
1000
900
Fluorescence Intensity
800
700
600
500
400
300
200
100
0
0
2
4
6
8
10
12
Concentration (ppm)
Figure 4.26: Calibration graph for functionalized ZnS binding with protein lysozyme
under optimum conditions. pH 7, [Functionalized ZnS colloids] = 4x10-4 mol L-1.
y = 21.911x + 479.13
1200
R2 = 0.9367
Fluorescence Intensity
1000
800
600
400
200
0
0
5
10
15
20
25
Concentration (ppm)
Figure 4.27: Calibration graph for functionalized ZnS binding with protein egg
albumin under optimum conditions. pH 7, [Functionalized ZnS colloids] = 4x10-4
mol L-1.
97
4.11
Conclusions
The fluorescence method of functionalized ZnS binding with BSA has been
successfully developed. The results have led to some interesting phenomena. An
optimum pH 7, temperature 25 oC and reaction time 20 min was obtained for BSA
determination. Besides, under optimum condition, the fluorescence intensity is
proportional to the concentration of BSA in the linear range of 0.4 ppm to 22.5 ppm.
The corresponding detection limit is 0.09 ppm. In brief, the application of
functionalized ZnS binding with BSA as fluorescence probe for protein leads to a
particularly sensitive, stable, simple and selective method.
CHAPTER 5
CONCLUSIONS AND SUGGESTIONS
5.1
Conclusions
This study involved a development of CdS capped mercaptoacetic acid
(functionalized CdS) and ZnS capped cysteine (functionalized ZnS) for the assay of
protein.
The deep yellow colloid CdS capped mercapto acetic acid was used for the
binding with protein (BSA). The functionalized CdS have been covalently attached
to the protein due to the presences of capping agent mercapto acetic acid. The
fluorescence properties of functionalized CdS binding with protein were observed at
excitation wavelength Ȝexc of 233 nm wavelengths and emission wavelength (Ȝem) of
350 nm. The analysis of the functionalized CdS binding with protein was based on
the effect of fluorescence intensity enhancement when protein (BSA) interacted with
CdS capped mercaptoacetic acid.
The optimization procedures for the analysis of functionalized CdS binding
with protein were carried out in order to obtain a reliable method. The optimum pH 6
and Tris buffer was adopted for the analysis and the maximum fluorescence intensity
occurred when the functionalized CdS colloid concentration was in the range 3.0 x
10-4 mol L-1. The influence of the reaction time and the temperature on CdS binding
with protein showed that the fluorescence intensity reached maximum and constant
value when the solution was incubated for 20 min at room temperature 25 oC.
99
The standard calibration curve for the quantitative analysis of protein binding
with functionalized CdS showed R2 value 0.9899, regression equation y = 10.56x +
393.89 and the linearity range was between 2-50 ppm. The 3ı limit of the detection
of the developed assay is 0.14 ppm. The results obtained from 10 independent runs
of the BSA were reproducible with average RSD of 1.5 %.
The salt dependence study on the functionalized CdS binding with BSA
clearly demonstrated that the binding is due to nonelectrostatic interaction. The
interferences of amino acids, glucose, starch and metal ions showed that the
developed method has high selectivity and it was applied to the determination of
various proteins such as egg albumin, lysozyme and amlayese under the optimum
condition. The standard calibration graph for the various protein determinations
illustrated R2 is in the range of 0.9722 to 0.9897.
Besides functionalized CdS, the study of ZnS capped cysteine (functionalized
ZnS) was employed for the binding with BSA. Through the surface modification of
the ZnS by cysteine, the sensitive fluorescence method for assay of protein had been
developed. In this investigation, the maximum emission fluorescence peak of
functionalized ZnS is located at 357 nm (Ȝexc = 233 nm). It was found out that the
emission maxima of the fluorescence of functionalized ZnS and BSA remained at
similar wavelength but the intensities are significantly enhanced. The enhancement
of emission intensity of the mixture of functionalized ZnS and BSA suggested that
interaction of functionalized ZnS binding with BSA had taken place. The optimum
pH 7 with Tris buffer and reaction time 20 min under the room temperature was
found to be suitable in this protein binding assay. The constant emission intensity
was obtained at concentration of functionalized ZnS in range of 4.0 x 10-4 mol L-1.
Under the optimum conditions, calibration graphs were plotted with
fluorescence intensity against the various concentration of BSA. The fluorescence
intensity is directly proportional to the concentration of BSA with linear range of 0.4
to 22.5 ppm and the correlation coefficient is R2 = 0.9805 with y = 21.886x + 456.05.
This result indicates that the developed assay is useful for the quantitative analysis of
protein. The 3ı limit of the detection of the developed assay is 0.09 ppm and RSD
value with 1.7% suggested the developed assay is sensitive and reproducible. Based
100
on the study of the influence of various ions and non-protein substances on the BSA
determination, it is widely accepted that the developed assay is very selective due to
the little effect on the BSA determination. The method was employed to various
types of protein such as egg albumin, lysozyme and amylase. The calibration graphs
for the various proteins were constructed showed R2 value > 0.9800. It can be
concluded that the developed protein assay functionalized ZnS binding BSA leads to
a particularly sensitive, stable, simple and selective method.
In conclusion, generally, the application of CdS capped mercaptoacetic acid
(functionalized CdS) and ZnS capped Cysteine (functionalized ZnS) for the binding
with BSA was compared. It is generally agreed that the fluorescence characterization
and the optimization results showed a similar results, however few comparison was
made in term of the stability of the functionalized ZnS and functionalized CdS. The
reaction time of functionalized ZnS binding with BSA retained for 5 hours compared
to functionalized CdS, which was retained for 3 hours. It is generally agreed that the
stability of functionalized ZnS binding with BSA are more stable because the
intensity remains constant for 5 h.
Comparison using mercaptoacetic acid as stabilizer, cysteine is less expensive
and nontoxic. The salt dependence study showed that the binding modes between the
functionalized CdS with BSA group are mainly due to non-electrostatic binding
meanwhile the binding between ZnS may be due to the hydrophobic binding.
Moreover, the influences of non-protein substances mainly amino acids affect the
BSA binding with functionalized ZnS compared to the functionalized CdS. This may
due to the presence of cysteine as capping agent from the same group of amino acid
compound. The limit of the detection (LOD) of functionalized ZnS is 0.09 ppm,
which is slightly more sensitive compared to functionalized CdS with 0.14 ppm.
By comparison with fluorescence labeling method utilizing organic dyes, the
developed functionalized CdS and functionalized ZnS as protein (BSA) assay have a
wider linear range larger Stoke’s shift. The sensitivity of organic dye binding assay
showed a similar value of limit of detection with the developed assay. In addition,
the organic dyes are more expensive compared to inorganic compound CdS and ZnS.
The organic dye binding protein methods have limitations such as narrow excitation
101
bands and broad emission bands with red spectral tails and spectral overlap. Most of
the organic dyes binding with protein exhibited low resistance to photobleaching. In
a word, the luminescent colloidal semiconductor CdS and ZnS have the potential to
overcome problem in labeling protein application by combining the advantages of
high photobleaching, good chemical stability and readily tunable spectral properties.
The development of protein assay based on the fluorescence enhancement
technique has achieved its objectives in this work with successful and promising
results. The application of the functionalized CdS and functionalized ZnS as
fluorescence probe for protein leads to a particularly sensitive, stable, simple and
selective assay.
5.2
Suggestions
Although overall promising results have been obtained in this work, there
was a need to complement some areas of the present study that were worth
investigating. Suggestions and ideas were proposed for future studies.
Further investigation should be conducted by capping the semiconductor with
other caping material with different kind of functional group such as –COOH, -OH,
-NH2 and -SH. In addition, the study of the labeling protein can be extended to the
production of other semiconductor compound such as CdSe, CuS and MnS.
For future work on the developing fluorescence probe, the assay was
proposed in other field such as chemical sensor, immune analysis and other
biotechnology applications which their application still unclear.
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