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STUDYING THE INTERACTION BETWEEN BIOMACROMOLECULES AND SMALL MOLECULES INCLUDING DRUGS, DYES AND NANOSTRUCTURES

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Ph.D. Dissertation in Analytical Chemistry

Studying the Interaction between Biomacromolecules AND Small Molecules Including Drugs, Dyes and Nanostructures

ABSTRACT

The interactions of different compounds, with bovine serum albumin (BSA), human serum albumin (HSA), or calf thymus DNA (CT-DNA) have been studied by spectroscopic techniques such as fluorescence and UV–Visible absorption spectroscopy at physiological condition (pH 7.40). The quenching constants and binding parameters (binding constants and number of binding sites) were determined by fluorescence quenching method. The experiments were done at different temperatures. The thermodynamic parameters were calculated (ΔG⁰, ΔH⁰ and ΔS⁰), then the interaction forces of the reaction were determined from sign and magnitude of the thermodynamic parameters. The displacement experiments with site markers were done and the binding sites of the ligands were obtained. Moreover, the conformational changes of protein were revealed by synchronous fluorescence and UV-Vis spectroscopy. Finally, the distance, r, between donor (serum albumin) and acceptor (ligands) were obtained according to Förster theory of non-radiation energy transfer.

A simple analytical method based on the second-order calibration of the pH gradient spectrophotometric data was developed for assay of carminic acid (CA) in human plasma and orange. The parallel factor analysis (PARAFAC) was coupled with standard addition to encounter the significant effects of plasma and juices matrices on the acid-base behavior and UV-Vis absorbance spectra of CA. The results confirmed the success of the proposed method in the analysis of pH gradient spectrophotometric data for determination of CA.

A new method for the determination of vitamin B12 based on the fluorescence quenching of BSA-stabilized gold nanoclusters (BSA-AuNCs) was developed. The fluorescence emission was measured at λ_ex/λ_em 370/617 nm in phosphate buffer solution (pH 7.4), and the experimental variables and possible interferences were studied. Thus, a simple, sensitive, and selective method for rapid determination of vitamin B12 was described. The proposed method was successfully applied to commercial injection ampoules.

دسته: زبان خارجه, زبان خارجه برچسب: DYES AND NANOSTRUCTURES, STUDYING THE INTERACTION BETWEEN BIOMACROMOLECULES AND SMALL MOLECULES INCLUDING DRUGS

توضیحات

TABLE OF CONTENTS

Contents…………………………………………………………………………………………………. Page

CHAPTER ONE INTRODUCTION.. 1

1.1 Serum Albumin. 2

1.1.1 Structure and Properties of Serum Albumin. 2

1.1.2 UV/Vis Spectra of Serum Albumin. 4

1.1.3 Fluorescence Spectra of Serum Albumin. 5

1.1.4 Protein-Ligand Interaction. 7

1.1.5 Binding Sites of Serum Albumin. 8

1.1.6 Type of Interaction Force between Ligands and Serum Albumin. 9

1.2 DNA.. 10

1.2.1 DNA Structures. 10

1.2.2 Ultraviolet Absorption Spectra of DNA.. 12

1.2.3 Fluorescence Spectra of DNA.. 12

1.2.4 DNA-Drug Interaction. 13

1.2.5 Binding Mode of Drugs – DNA.. 14

1.2.5.1 Covalently Bonding. 14

1.2.5.2 Non-Covalently Bonding. 15

1.3 Techniques Used to Study the Interaction between Biomacromolecules with Different Ligands 17

1.3.1 UV/Vis Absorption Spectroscopy. 17

1.3.2 Fluorescence Spectroscopy. 18

1.3.2.1 Quenching of Fluorescence. 19

1.3.2.2 Stern Volmer Plot 21

1.3.2.3 Binding Parameter 22

1.3.2.4 Modified Stern-Volmer Plots. 23

1.3.2.5 Fluorescence Resonance Energy Transfer (FRET) 25

1.3.2.6 Synchronous Fluorescence Spectroscopy Studies. 26

1.3.3 Infrared Spectroscopy. 27

1.4 Harmalol 28

1.5 Amodiaquine (AQ) 29

1.6 Metal-Containing Drugs. 30

1.7 Ethidium Bromide. 32

1.8 Cyanine Dyes. 33

1.9 Quantum Dots. 34

1.10 Carminic Acid. 35

1.11 Vitamin B12. 36

1.12 Gold Nanoclusters. 37

CHAPTER TWO LITERATURE REVIEW… 39

2.1 Interaction of Serum Albumin with Different Ligands. 40

2.1.1 Studies on the Interaction between Harmalol and Serum Albumin. 44

2.1.2 Investigation on the Interaction between Amodiaquine and Serum Albumin 45

2.1.3 Study of the Interaction of Anticancer Pt(II) Complexes with Serum Albumins 45

2.1.4 Study of the Interaction of Anticancer Pt(II) Complexes with DNA.. 47

2.1.5 Study of the Interaction of Cyanine Dyes with Serum Albumins. 48

2.1.6 Study on the Interaction between Serum Albumin (SA) and QDs. 49

2.2 Determination of Carminic Acid. 50

2.3 Detemination of Vitamin B12. 51

2.4 Objective of the Present Work. 52

CHAPTER THREE EXPERIMENTAL.. 53

3.1 Interaction of Different Ligands with Biomacromolecules. 54

3.1.1 Materials. 54

3.1.2 Apparatus. 54

3.1.3 Fluorescence Spectroscopy Studies on the Interaction between Harmalol and Human Serum Albumin 55

3.1.3.1 Preparation of Solution. 55

3.1.3.2 Fluorescence Titration Experiments. 56

3.1.3.3 FT-IR Measurement 56

3.1.4 Investigation of the Interaction between Amodiaquine and Human Serum Albumin by Fluorescence Spectroscopy. 57

3.1.4.1 Preparation of Solutions. 57

3.1.4.2 Spectroflourimetric Experiments. 57

3.1.4.2.1 Binding Assay. 57

3.1.4.2.2 Site Marker Competitive Experiments. 58

3.1.5 Affinity of Two Novel Five-Coordinated Anticancer Pt(II) Complexes to Human and Bovine Serum Albumins. 58

3.1.5.1 Preparation of Solution. 58

3.1.5.2 Procedures. 59

3.1.5.2.1 Fluorescence Measurements. 59

3.1.5.2.2 Site Marker Competitive Experiments. 60

3.1.5.2.2.1 Warfarin as Marker of Site I. 60

3.1.5.2.2.2 Ibuprofen as Marker of Site II. 61

3.1.6 Study on the Interaction Mechanism Between DNA and Two Newly Five-Coordinated Anticancer Pt(II) Complexes. 61

3.1.6.1 Preparations of Solutions. 61

3.1.6.2 Absorption Measurements. 62

3.1.6.3 Fluorescence Measurements. 62

3.1.6.3.1 Fluorescence Enhancement Experiments. 62

3.1.6.3.2 Competitive Binding Studies. 63

3.1.6.4 Viscosity Measurements. 63

3.1.6.5 Differential Scanning Calorimetry. 64

3.1.7 Investigation of the Interaction between Merocyanine Dye and Bovine Serum Albumin 64

3.1.7.1 Preparation of Solution. 64

3.1.7.2 Fluorescence Measurements for Binding Study. 65

3.1.7.3 UV–Visible Measurements for Binding Study. 66

3.1.8 Study of the Interaction between Human Serum Albumin and ZnS:Mn Quantum Dots 66

3.1.8.1 Synthesis of ZnS:Mn Quantum Dot 66

3.1.8.2 Preparation of Solutions. 67

3.1.8.3 Spectroflourimetric Experiments. 67

3.2 Spectrophotometric Determination of Carminic Acid in Human Plasma and Fruit Juices 68

3.2.1 Chemicals. 68

3.2.2 Apparatus. 69

3.2.3 Procedure. 69

3.2.4 Theory of PARAFAC in Spectral-pH Absorbance Data Matrices. 70

3.3 Using of BSA-modified Au nanoclusters for Determination of Vitamin B12 in Pharmaceutical Preparations. 72

3.3.1 Reagents. 72

3.3.2 Apparatus and Procedure. 73

3.3.3 Synthesis of BSA-Modified Gold Nano Cluster 73

3.3.4 Procedure. 73

CHAPTER FOUR RESULTS AND DISCUSSION.. 75

4.1 Fluorescence Spectroscopy Studies on the Interaction between Harmalol and Human Serum Albumin 76

4.1.1 Fluorescence Quenching Mechanism.. 76

4.1.2 Determinations of Binding Constants and Number of Binding Sites. 79

4.1.3 Thermodynamic Analysis and the Nature of the Binding Force. 80

4.1.4 The Conformation Study of HSA upon Binding with Harmalol 82

4.1.4.1 Synchronous Fluorescence Spectroscopy. 82

4.1.4.2 FT-IR Measurement 83

4.1.5 Confirming the Binding Sites on HSA.. 84

4.1.6 Distance Measurement between Tryptophan and the Harmalol Binding Site on HSA 86

4.1.7 Conclusions. 86

4.2 Investigation of the Interaction between Amodiaquine and Human Serum Albumin 87

4.2.1 Fluorescence Quenching Measurements. 88

4.2.2 Analysis of Binding Equilibria. 90

4.2.3 Thermodynamic Parameters for Binding of AQ to HSA.. 92

4.2.4 Identification of Binding Site of AQ on HSA.. 93

4.2.5 Investigation of HSA Conformation Changes. 95

4.2.6 Energy transfer between AQ and HSA.. 96

4.2.7 Conclusion. 96

4.3 Affinity of Two Novel Five-Coordinated Anticancer Pt(II) Complexes to Human and Bovine Serum Albumin. 100

4.3.1 The Effect of HSA/BSA on the Fluorescence Emission of Pt complexes. 101

4.3.2 Fluorescence Quenching Studies. 103

4.3.3 Binding Parameters. 111

4.3.4 Thermodynamic Parameters and Binding Mode. 112

4.3.5 Site-Selective Binding of Pt Complexes on HSA/BSA.. 114

4.3.6 Synchronous Fluorescence Spectroscopy Studies. 116

4.3.7 Energy Transfer between Pt Complexes and SA.. 118

4.3.8 Conclusions. 120

4.4 Study on the interaction mechanism between DNA and two newly Five-Coordinated Anticancer Pt(II) Complexes. 121

4.4.1 Native Fluorescence of Complexes. 123

4.4.2 EB-DNA Quenching Assay. 124

4.4.3 UV-Spectral Study. 127

4.4.4 Viscosimetric Studies. 128

4.4.5 Differential Scanning Calorimetry Results. 129

4.4.6 Conclusions. 130

4.5 The Investigation of the Interaction between Cyanine Dye and Bovine Serum Albumin 131

4.5.1 Fluorescence Quenching Studies. 131

4.5.2 Type of Interaction Forces between Dye and BSA.. 134

4.5.3 Identification of the Binding Site of Dye on BSA.. 135

4.5.4 Energy Transfer from BSA to Dye. 137

4.5.5 Synchronous Fluorescence Spectroscopic Studies of Dye-Bound BSA.. 138

4.5.6 Absorption Spectroscopy. 140

4.5.7 Conclusions. 141

4.6 Interaction between Human Serum Albumin and ZnS:Mn Quantum Dots. 142

4.6.1 Characterization of ZnS quantum dots. 142

4.6.1.1 TEM images of the quantum dots. 142

4.6.1.2 X-ray diffraction (XRD) 143

4.6.1.3 Optical characteristics of ZnS:Mn nanoparticles. 144

4.6.2 The Influence of ZnS:Mn QDs Concentration on the Fluorescence Intensity of HSA 145

4.6.3 Thermodynamic Parameters and Binding Mode. 149

4.6.4 Binding Parameter 151

4.6.5 HSA Conformation Changes. 152

4.6.5.1 UV Absorption Spectra of the System.. 152

4.6.5.2 Synchronous Fluorescence Spectroscopic Studies. 153

4.6.6 Conclusions. 157

4.7 Spectrophotometric Determination of Carminic Acid in Human Plasma and Fruit Juices 157

4.7.1 Calibration in Buffer Solutions. 160

4.7.2 Determination of CA in Human Plasma by PARAFAC.. 166

4.7.3 Determination of CA in Orange Juice by PARAFAC.. 173

4.7.4 Conclusion. 177

4.8 Using of BSA-modified Au nanoclusters for Determination of Vitamin B12 in Pharmaceutical Preparations. 178

4.8.1 Characterization of BSA-AuNC.. 179

4.8.2 The Fluorescence Quenching of BSA-AuNC by Vitamin B12. 181

4.8.3 Effect of pH and Buffer Solution. 182

4.8.4 Method Validation for Vitamin B12 Quantification. 184

4.8.5 Effect of Foreign Substances. 184

4.8.6 Determination of Vitamin B12 in Pharmaceutical Preparations. 185

4.8.7 Conclusions. 186

REFERENCES. 187

List of Figures

Contents Page

Figure ‎1.1.1 Schematic representation of: (a) tertiary structure; and, (b) secondary structure of HSA. 4

Figure ‎1.2.1 Chemical structure of nucleic acid constituents, the normal base (a) and sugars (b). 11

Figure ‎1.2.2 Mechanistic pathway for DNA functionalization by interstrand cross-linking agent. A and B represent electrophilic moieties within the cross-linking agent of interest. 15

Figure ‎1.4.1 The chemical structure of harmalol. 29

Figure ‎1.5.1 The chemical structure of amodiaquine. 30

Figure ‎1.6.1 Structures of cisplatin (a), carbopatin (b), and oxaliplatin (c). 31

Figure ‎1.7.1 Chemical structures of EB. 32

Figure ‎1.10.1 The chemical structure of carminic acid. 36

Figure ‎1.11.1 The chemical structure of vitamin B12. 37

Figure ‎3.1.1 The chemical structure of Pt complexes. 59

Figure ‎3.1.2 The chemical structure of dye. 65

Figure ‎4.1.1 Fluorescence spectra of HSA with harmalol at 298 K (solid lines). The concentrations of HSA was 1.0 × 10⁻⁶ mol L⁻¹; harmalol: (a) 0.0, (b) 1.3, (c) 2.0, (d) 2.7, (e) 3.7, (f) 4.7, (g) 5.7, (h) 6.7, (i) 8.3, (j) 10.0, (k) 11.7, (l) 13.3 ×10⁻⁶ mol L⁻¹. The dotted line is the spectra of aqueous solution of harmalol (13.3 ×10⁻⁶ mol L⁻¹) in pH 7.4. For all spectra λ_exis 280 nm. 77

Figure ‎4.1.2 Stern-Volmer plot for quenching HSA with harmalol in buffer solution at 292 K (●), 298 K (Δ), 305 K (■) and 310 K (◊). 78

Figure ‎4.1.3 The modified Stern–Volmer plots of HSA on the different temperature with harmalol. λex = 280 nm (292 K (●), 298 K (Δ), 305 K (■) and 310 K (◊)). 80

Figure ‎4.1.4 Double-log plots of harmalol quenching effect on HSA fluorescence at T=298K. 81

Figure ‎4.1.5 Van’t Hoff plot for the interaction of HSA and harmalol in Tris buffer, pH 7.40. 82

Figure ‎4.1.6 The synchronous fluorescence spectra of HSA–harmalol. (A) Δλ= 60 nm; (B) Δλ= 15 nm C_HSA= 1.0 × 10⁻⁶ mol L⁻¹ while concentrations of harmalol were changed from 0.00 to 13.3 ×10⁻⁶. 83

Figure ‎4.1.7 The infrared spectra (1500–1700 cm^-1) before (solid line) and after protein interacting with harmalol (dashed line) at the harmalol to protein molar ratio of 1 (C_HSA = C_harmalol = 1.0× 10⁻⁴ mol L⁻¹) Tris-HCl buffer solution (pH 7.4). 84

Figure ‎4.1.8 The overlap of fluorescence spectra of HSA (a) with absorbance spectra of harmalol (b): C_HSA = C_harmalol = 1.0× 10⁻⁶ mol L⁻¹. 86

Figure ‎4.2.1 Fluorescence spectra of HSA with AQ at 291 K. The concentrations of HSA was 1.1 × 10⁻⁶ mol L⁻¹; AQ: (a) 0.0, (b) 0.7, (c) 1.3, (d) 2.0, (e) 3.0, (f) 4.0, (g) 5.3, (h) 6.7, (i) 8.3, (j) 10.0, (k) 12.3, (l) 15.0 ×10⁻⁶ mol L⁻¹. λ_ex: 280 nm. 88

Figure ‎4.2.2 Stern-Volmer plot for quenching HSA with AQ in buffer solution at 291 K (●), 301 K (○) and 310 K (▲).pH 7.4 and λ_ex: 280 nm. 89

Figure ‎4.2.3 The modified Stern–Volmer plots of HSA on the different temperature with AQ at 291 K (●), 301 K (○) and 310 K (▲).pH 7.4 and λ_ex: 280 nm. 90

Figure ‎4.2.4 Double-log plots of AQ quenching effect on HSA fluorescence at 291 K (●), 301 K (○) and 310 K (▲).pH 7.4 and λ_ex: 280 nm. 91

Figure ‎4.2.5 Van’t Hoff plot for the interaction of HSA and AQ in Tris buffer, pH 7.40. 92

Figure ‎4.2.6 Effect of site marker to the AQ-HSA system (T = 291 K). (A) C_warfarin= C_HSA = 1.1 × 10⁻⁶ mol L⁻¹ (λ_ex = 280 nm); (B) C_Ibuprofen = C_HSA = 1.1 × 10⁻⁶ mol L⁻¹ (λ_ex = 280 nm); C_AQ/( 10⁻⁶ mol L⁻¹), a-l: 0.0, 0.7, 1.3, 2.0, 3.0, 4.0, 5.3, 6.7, 8.33, 10.0, 12.3, 15.0. (C) Titration of warfarin–HSA by AQ with concentrations from 0 to 1.5×10⁻⁵ mol L⁻¹, (a) to (j); curve k shows the emission spectrum of warfarin only. C_warfarin= C_HSA = 1.1 × 10⁻⁶ mol L⁻¹, λ_ex=320 nm. 98

Figure ‎4.2.7 (A)The UV-vis absorption spectra of HSA in the absence and presence AQ. Solid line, the absorption spectrum of HSA only and dash line, the absorption spectrum of HSA in the presence of AQ at the same concentration, C_HSA = C_AQ = 4.0×10⁻⁶ mol L⁻¹; (B,C)The synchronous fluorescence spectra of HSA–AQ. (B) Δλ= 60 nm; (C) Δλ= 15 nm that C_HSA = 1.1 × 10⁻⁶ mol L⁻¹ while concentrations of AQ were from 0.0 to 15.0 ×10⁻⁶ mol L⁻¹. 99

Figure ‎4.2.8 The overlap of the fluorescence spectra (a) of HSA and the absorbance spectra (b) of AQ, C_HSA = C_AQ = 1.1 µM (301 K). 100

Figure ‎4.3.1 The fluorescence emission spectra of 1 (left) and 2 (rigth) with HSA (up) and BSA (down); (a) 5.0 × 10⁻⁶ mol L⁻¹ Pt complexes; (b)–(i) 5.0 × 10⁻⁶ mol L⁻¹ Pt complexes in the presence of 0.02, 0.05, 0.1, 0.2, 0.7, 1.3, 2.6, 6.2 × 10⁻⁶ mol L⁻¹ SA. 103

Figure ‎4.3.2 Changes in the fluorescence spectra of HSA (up (A)) and BSA (down (B)) through their titration with 1 (left (1)) and 2 (right (2)) at 300 K. The concentration of both proteins is 1.3 × 10⁻⁶ mol L⁻¹ and Pt(II) complexes concentration was varied from (a) 0.0 to (j) 5.0 × 10⁻⁶ mol L⁻¹; pH 7.4 and λ_ex: 280 nm. 104

Figure ‎4.3.3 Stern-Volmer plot for quenching of different Pt(II) complexes: 1 (1) and 2 (2) to HSA (A) and BSA (B) at 300 K (□) and 310 K (■). 105

Figure ‎4.3.4 The UV-vis absorption spectra of HSA (top) and BSA (down) in the absence and presence of 1 (left) and 2 (rigth). Solid line: the absorption spectrum of proteins and dash line: the absorption spectrum of proteins in the presence of Pt complexes at the same concentration, C_HSA = C_{Pt complexes} = 3.5×10⁻⁶ mol L⁻¹. The inset shows the enlarged spectra in the wavelength range of 250-300 nm. 108

Figure ‎4.3.5. Changes in the fluorescence spectra of HSA upon titration with 1 (A (1)) and 2 (A (2)) at 300 K. The concentration of HSA is 1.3 × 10⁻⁶ mol L⁻¹ and Pt(II) complexes concentration was varied from (a) 0.0 to (g) 5.0 × 10⁻⁶ mol L⁻¹; pH 7.4 and λ_ex: 280 nm. 109

Figure ‎4.3.6 The modified Stern–Volmer plots of HSA (A) and BSA (B) on the different temperature for 1 (1) and 2 (2). λ_ex = 280 nm ( 300 K (□) and 310 K (■)); pH = 7.4. 110

Figure ‎4.3.7 Double-log plots of 1 (1) and 2 (2) quenching effect on HSA (A) and BSA (B) fluorescence at 300 K (□) and 310 K (■). 112

Figure ‎4.3.8 Effect of 1 (1) and 2 (2) complexes to warfarin-HSA (A) and warfarin-BSA (B) system (λ_ex=320 nm). a–g: C_warfarin = C_SA =1.3 × 10^-6 mol L⁻¹, 1 and 2 concentration was: (a) 0.0, (b) 0.8, (c) 1.7, (d) 2.5, (e) 3.3, (f) 4.2, (g), 5.0(h) 6.7, (i) 8.3 and (j) 11.7 ×10⁻⁶ mol L⁻¹; curve k shows the emission spectrum of warfarin only. 115

Figure ‎4.3.9 Synchronous fluorescence spectra of HSA (A) and BSA (B) (1.3 × 10⁻⁶ mol L⁻¹) upon addition of 1 (1) and 2 (2); Δλ= 60 nm. The concentration of Pt complexes from a−j was 0.0 − 5.0×10⁻⁶ mol L⁻¹. 117

Figure ‎4.3.10 Synchronous fluorescence spectra of HSA (A) and BSA (B) (1.3 × 10⁻⁶ mol L⁻¹) upon addition of 1 (1) and 2 (2); Δλ= 15 nm. The concentration of Pt complexes from a−j was 0.0 − 5.0 ×10⁻⁶ mol L⁻¹. 118

Figure ‎4.3.11 The overlap of the fluorescence spectra (a) of HSA (A) and BSA (B) and the absorbance spectra (b) of 1 (1) and 2 (2) complexes, C_SA = C_{Pt complex} = 1.3 µM (300 K). 119

Figure ‎4.4.1 The fluorescence emission spectra of 1 (A) and 2 (B) with the addition of CT-DNA; 1.0 × 10⁻⁵ mol L⁻¹ Pt(II) complexes in the absence (a) and presence of 1.8, 6.0, 12.3, 19.3, 29.5, 42.8, 59.0 and 89.8 × 10⁻⁶ M DNA (b to i, respectively); pH = 7.4 and λ_ex = 320 nm. 124

Figure ‎4.4.2 Variations in fluorescence intensity of 3.9×10⁻⁶ mol L⁻¹ EB in the presence of increasing concentration of DNA (0.0, 7.0, 15.0, 25.0, 35.0, 45.0 and 55.0×10⁻⁶ mol L⁻¹). 125

Figure ‎4.4.3 Fluorescence spectra of EB bound to DNA in the absence and presence of Pt complexes 1 (A) and 2 (B). C_EB = 3.9×10⁻⁶ mol L⁻¹; C_DNA = 25.0×10⁻⁶ mol L⁻¹; C_complex = 0.0, 2.5, 5.0, 7.5, 10.0, 13.0, 16.0, 20.0, 25.0, 30.0, 35.0 and 40.0×10⁻⁶ mol L⁻¹for curves a–l, respectively; pH = 7.4; λ_ex = 525 nm. The inset shows the Stern-Volmer plot. 126

Figure ‎4.4.4 Absorption spectra of 1 (A) and 2 (B) in the presence of DNA at different concentrations. C_complex= 50.0×10⁻⁶ mol L⁻¹and C_DNA = 0.0, 1.2, 2.4, 4.8, 7.2 and 9.6×10⁻⁶ mol L⁻¹(curves a-f, respectively). 128

Figure ‎4.4.5 Effect of increasing amounts of Pt(II) complexes 1 (■) and 2 (□) on the relative viscosity of CT-DNA in 0.05 mol L⁻¹ phosphate buffer solution of pH 7.4. C_DNA = 50 µM. 129

Figure ‎4.4.6 The DSC curves of DNA (a), DNA with ligand 1 (b), and DNA with ligand 2 (c). 130

Figure ‎4.5.1 Fluorescence spectra of BSA with dye at 300 K. The concentrations of BSA was 1.0 × 10⁻⁶ mol L⁻¹; dye: (a) 0.0, (b) 1.1, (c) 2.3, (d) 3.4, (e) 4.5, (f) 5.7, (g) 7.4, (h) 9.1, (i) 11.3, (j) 14.2, (k) 17.0 ×10⁻⁶ mol L⁻¹ (l) only dye with concentration 22.7 ×10⁻⁶ mol L⁻¹. pH 7.4 and λ_ex: 280 nm. 132

Figure ‎4.5.2 Stern-Volmer plot for quenching BSA with dye in buffer solution at 290 K (●), 300 K (Δ) and 308 K (■). 133

Figure ‎4.5.3 The modified Stern–Volmer plots of BSA on the different temperature with dye. λ_ex = 280 nm (at 290 K (●), 300 K (Δ) and 308 K (■)). 134

Figure ‎4.5.4 Van’t Hoff plot for the interaction of BSA and dye in Tris buffer, pH 7.4. 135

Figure ‎4.5.5 Double-log plots of dye quenching effect on BSA fluorescence at 290 K (●), 300 K (Δ) and 308 K (■). 136

Figure ‎4.5.6 The overlap of the fluorescence spectra (a) of BSA and the absorbance spectra (b) of dye, c(BSA) = c(dye) = 1.0 µM (300 K). 138

Figure ‎4.5.7 The synchronous fluorescence spectra of BSA–dye. (A) Δλ= 15 nm; (B) Δλ= 60 nm. C_BSA = 1.0 × 10⁻⁶ mol L⁻¹ while concentrations of dye were from 0.0 to 17.0 ×10⁻⁶ mol L⁻¹. 139

Figure ‎4.5.8 The UV-vis absorption spectra of dye (2.0 ×10⁻⁵mol L⁻¹) in the absence and presence of BSA (from 0.0 to 5.8 ×10⁻⁵ mol L⁻¹). 141

Figure ‎4.6.4 Changes in the fluorescence spectra of HSA through their titration with ZnS:Mn 1% (a), ZnS:Mn 2% (b) and ZnS:Mn 1% (c) at 291 K. The concentration of HSA is 1.0 × 10⁻⁶ mol L⁻¹ and ZnS Qds concentration was varied from (a) 0.0 to (j) 10.0 × 10⁻⁶ mol L⁻¹; pH 7.4 and λ_ex: 280 nm. 146

Figure ‎4.6.5 Stern-Volmer plot for quenching of different ZnS QDs: 1% Mn (a), 2% Mn (b) and 3% Mn (c) to HSA at 291 K (●), 298 K(Δ) and 300 K (■). 148

Figure ‎4.6.6 The modified Stern–Volmer plots for quenching of different ZnS QDs: 1% Mn (a), 2% Mn (b) and 3% Mn (c) to HSA at 291 K (●), 298 K (Δ) and 308 K (■) pH = 7.4. 150

Figure ‎4.6.7 Van’t Hoff plot for the interaction of HSA and different ZnS QDs: 1% Mn (●), 2% Mn (Δ) and 3% Mn (■)in phosphate buffer, pH 7.4. 151

Figure ‎4.6.8 Double-log plots of different ZnS QDs: 1% Mn (●), 2% Mn (Δ) and 3% Mn (■) quenching effect on HSA fluorescence at 291 K. 152

Figure ‎4.6.9 UV–Vis absorption spectra of HSA (1.0×10⁻⁶ mol L⁻¹) in the presence of 0.0–6.7×10⁻⁶ mol L⁻¹ of ZnS with 1% Mn (a), 2% Mn (b) and 3% Mn (c). 154

Figure ‎4.6.10 Synchronous fluorescence spectra of HSA (1.0 × 10⁻⁶ mol L⁻¹) in the presence of 0.0–10.0×10⁻⁶ mol L⁻¹ of ZnS with 1% Mn (a), 2% Mn (b) and 3% Mn (c).; Δλ= 60 nm. 155

Figure ‎4.6.11 Synchronous fluorescence spectra of HSA (1.0 × 10⁻⁶ mol L⁻¹) in the presence of 0.0–10.0×10⁻⁶ mol L⁻¹ of ZnS with 1% Mn (a), 2% Mn (b) and 3% Mn (c).; Δλ= 15 nm. 156

Figure ‎4.7.1 Changing in absorbance spectra of CA in buffer solution as function of pH. 161

Figure ‎4.7.2 Loadings of the PARAFAC model with four factors built with calibration samples. (a) pH mode, (b) wavelength mode and (c) concentration mode. 165

Figure ‎4.7.3 pH dependence of the spectral features of carminic acid in serum measured by UV/Vis absorption spectroscopy. 167

Figure ‎4.7.4 pftest plots for determination of number of factors in PARAFAC for serum sample. 170

Figure ‎4.7.5 Loadings of the PARAFAC model with two factors built for serum sample. (a) pH mode, (b) wavelength mode and (c) concentration mode. 173

Figure ‎4.7.6 pH dependence of the spectral features of carminic acid in orange juice measured by UV/Vis absorption spectroscopy. 174

Figure ‎4.7.7 pftest plots for determination of number of factors in PARAFAC for orange juice sample. 175

Figure ‎4.7.8 Loadings of the PARAFAC model with two factors built for orange juice sample. (a) pH mode, (b) wavelength mode and (c) concentration mode. 177

Figure ‎4.8.1 TEM images of BSA-AuNCs. Scale bar is 25 nm. 180

Figure ‎4.8.2 Absorption spectra of BSA-AuNCs (dashed line), fluorescence spectra (excited at 370 nm) of BSA-AuNCs (solid line) and absorption spectra of vitamin B12 (dotted line). 180

Figure ‎4.8.3 Fluorescence spectra of the BSA-AuNCs in the presence of increasing concentrations of vitamin B12. The arrows indicate the signal changes as increases in analyte concentrations 0.00, 0.16, 0.67, 1.33, 2.33, 3.32, 4.98, 7.28, 9.90, 13.16, 18.00, 24.39, 30.69 and 38.46 µg/ml. 182

Figure ‎4.8.4 The effect of pH on the measure of quenching. 183

Figure ‎4.8.5 The effect of buffer type on the measure of quenching. 183

Figure ‎4.8.6 Plots of the values of (F₀ – F)/F at 617 nm versus the concentration of vitamin B12. The excitation wavelength was set to 370 nm. The incubation time was 5 min. 184

List of Schemes

Contents Page

Table ‎1.1.1 Physical parameter of serum albumin. 3

Table ‎2.1.1 Selected Endogenous and Exogenous Ligands with Serum Albumin. 40

Table ‎2.1.2 Some reports on the interaction between Pt(II) complexes with SA. 46

Table ‎2.1.3 Some reports on the interaction between Pt(II) complexes with DNA. 47

Table ‎2.1.4 Some reports on the interaction cyanine dye with SA. 48

Table ‎2.1.5 Some reports on the interaction QD with SA. 49

Table ‎4.1.1 Stern-Volmer quenching constants (K_SV) and bimolecular quenching rate constant (k_q) of the interaction of harmalol with HSA at different temperatures. 77

Table ‎4.1.2 Modified Stern-Volmer association constant K_a and relative thermodynamic parameters at pH 7.40. 80

Table ‎4.2.1 Stern-Volmer quenching constants (K_SV) and bimolecular quenching rate constant (k_q) of the interaction of AQ with HSA at different temperatures. 89

Table ‎4.2.2 Modified Stern-Volmer association constant K_a and relative thermodynamic parameters at pH 7.40. 90

Table ‎4.2.3 Binding parameters of the system of the interaction AQ with HSA at different temperatures. 91

Table ‎4.2.4 Binding constants of competitive experiments of the AQ-HSA System (T = 291 K). 94

Table ‎4.3.1 Quenching Parameters of the interaction of Pt complexes with HSA/BSA at different temperatures. 105

Table ‎4.3.2 Modified Stern-Volmer association constant K_a and relative thermodynamic parameters of SA-Pt(II) complexes systems. 110

Table ‎4.3.3 Binding parameters of the system of the interaction Pt complexes with HSA/BSA at different temperatures. 111

Table ‎4.3.4 Förster energy transfer parameters of the interaction Pt complexes with HSA/BSA 120

Table ‎4.4.1 Stern-Volmer quenching constants (K_SV) and bimolecular quenching rate constant (k_q) of the interaction of Pt-complexes with DNA at 301 K. 127

Table ‎4.5.1 Stern-Volmer quenching constants (K_SV) and bimolecular quenching rate constant (k_q) of the interaction of cyanine dye with BSA. 133

Table ‎4.5.2 Modified Stern-Volmer association constant K_a and relative thermodynamic parameters at pH 7.4. 134

Table ‎4.5.3 Binding parameters of the system of the interaction dye with BSA at different temperatures. 136

Table ‎4.6.1 Quenching Parameters of the interaction QDs with HSA at different temperatures. 147

Table ‎4.6.2 Modified Stern-Volmer association constant K_a and relative thermodynamic parameters of HSA-QDs systems. 149

Table ‎4.7.1 Univariate calibration of CA in buffer solutions of various pHs. 164

Table ‎4.7.2 Dissociation constants of CA obtained from PARAFAC analysis of the three-way spectrophotometric data and those reported previouely. 166

Table ‎4.7.3 Results for the analysis of the unknown samples. 176

Table ‎4.8.1 Results of Determination of Vitamin B12 in Pharmaceutical Preparations. 186

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