Thymine Functionalized Silicon Quantum Dots for Selective Detection of Melamine and Hg2+

by

NAHIDA AKTER STUDENT ID.: 1015033102

MASTER OF PHILOSOPHY IN CHEMISTRY

DEPARTMENT OF CHEMISTRY

BANGLADESH UNIVERSITY OF ENGINEERING AND TECHNOLOGY

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CANDIDATE’S DECLARATION

It is hereby declared that this thesis or any part of it has not been submitted elsewhere for the award of any degree or diploma.

Nahida Akter

DEDICATION

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I dedicate this thesis to …..

My Beloved Family

&

Honorable supervisor

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Table of Contents

List of Tables and Figures ........................................................................................... vi

List of Abbreviations of Technical Symbols and Terms ............................................ ix

Acknowledgement …………………………………………………………...............x

Abstract ...................................................................................................................... xii

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List of Tables and Figures

Figures Figure 1.1. Quantum confinement effect in quantum dots ...................................................... 2

Figure 1.2. Principle of Stokes’s experiment………………………………………………....4

Figure 1.1. A brief illustration of the Jablonski diagram……………………………………..5

Figure 1.2. Absorption and emission spectra of Rhodamine 6G……………………...………6

Figure 1.3. Some common fluorophores……………………………………………………...7

Figure 1.4. A simplified Jablonski diagram……………………………………………..…....7

Figure 1.5. Aqueous solutions of the carbon dots…………………………………………….8

Figure 1.6. Important applications of GQDs and C-dots……………………………………...9

Figure 1.9. (a) SiQDs with visible emission (b) SiQDs with UV emission

(c) NIR/red emitting SiQDs………………………………………………………………….10

Figure 1.10. Colloidal synthesis of CdSe……………………………………………………12

Figure 1.11. Lithographic assembly…………………………………………………………13

Figure 1.12. Representative fluorescence and phase-contrast images of different cell types

labeled with CdS CQDs ……………………………………………………………….…….15

Figure 1.13. Schematic illustration of (a) QD−Apt (Dox) Bi-FRET system. (b specific uptake

of QD−Apt(Dox) conjugates into target cancer …………………………………..…………16

Figure 1.14. Types of fluorescence quenching………………………………………………19

Figure 1.15. Fluorescence Quenching of HTDC-QDs by Hg2+……………………………..21

Figure 1.16. Interaction of thymine with Hg2+ and melamine……………………………….24

Figure 1.17. SiQD-Thy based detection of melamine and Hg2+………...…………………..24

Figure 3.7. Reaction scheme for thymine functionalization of SiQDs…………………….. 45

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Figure 3.2. (a) FESEM image of SiQDs-Thy, (b) processed FESEM image of SiQDs-Thy

and (c) size distribution histogram after processing with MIPAR image analysis

software………………………………………………………………………………………46

Figure 3.3. Aqueous solution of SiQD-Thy under UV light (left) and day light (right)…….47

Figure 3.4. UV-vis absorption spectra of SiQDs and SiQDs-Thy………………………..…47

Figure 3.5. EDX spectrum of SiQDs-Thy…………………………………………………...48

Figure 8.6. XPS survey spectrum of SiQDs-Thy……………………………………………49

Figure 3.7. High resolution C1s spectra of SiQDs-Thy………………………………….….50

Figure 3.8. High resolution N1s spectra of SiQDs-Thy……………………………………..51

Figure 3.9. FTIR spectra of SiQDs and SiQDs-Thy………………………………………...52

Figure 3.10. Fluorescence emission spectra of SiQDs (a), SiQDs-Thy (b) ………...………54

Figure 9.11. UV-vis absorption (Abs), fluorescence excitation (Ex) and emission

(Em) spectra………………………………………………………………………………….55

Figure 3.12. (a) Fluorescence intensity changes of SiQDs-Thy in presence of different

concentration of Hg2+ (b) The plot of the fluorescence intensity ratio of SiQDs-Thy at 330 nm

versus the concentration of Hg2+ …………………………………………………………….56

Figure 3.13. Fluorescence intensity change of SiQDs-Thy with time in presence of

Hg2+…………………………………………………………………………………………..57

Figure 3.14. (a) Fluorescence intensity changes of SiQDs-Thy in the presence of different

concentration of melamine (b) The plot of the fluorescence intensity ratio of SiQDs-Thy at

330 nm versus the concentration of melamine……………………………………………….58

Figure 3.15. Fluorescence intensity change of SiQDs-Thy with time in presence

of melamine…………………………………………………………………………………..59

Figure 3.16. Selectivity study of SiQDs-Thy towards various metal ions and biomolecules

………………………………………………………………………………………………..59

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Tables

Table 3.1. Elemental composition obtained from the EDX analysis of SiQDs-Thy………...48

Table 3.2. Percentages of the elements obtained from the XPS survey spectrum …….……49

Table 3.3. Percentages of the bonds obtained from C 1s peak fitting ……………………....50

Table 3.4. Percentages of the bonds obtained from N 1s peak fitting ……………………....51

Table 3.5. Characteristic FTIR peaks and interpretations corresponding to SiQDS and

SiQDS-Thy………………………………………………………….. ……………………....53

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List of Abbreviations of Technical Symbols and

Terms

1. Quantum dots (QDs)

2. Silicon quantum dots (SiQDs)

3. Thymine functionalized silicon quantum dots (SiQDs-Thy)

4. (3‐ Aminopropyl)trimethoxysilane (APTMS)

5. N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC)

6. N-hydroxysuccinimide (NHS)

7. Fluorescence (FL)

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Acknowledgement At the very beginning, I humbly acknowledge my deepest gratitude to the almighty, the most

gracious, benevolent and merciful creator for his infinite mercy bestowed on me in carrying

out the research work presented in the dissertation.

It is a great pleasure for me to acknowledge my deepest sense of gratitude, sincere,

appreciation, heartfelt indebtedness and solemn regards to my reverend teacher and

supervisor Dr. Md. Shafiul Azam, Assistant Professor, Department of Chemistry, Bangladesh

University of Engineering and Technology (BUET), for his kind supervision, indispensable

guidance, valuable and constructive suggestions, liberal help and continuous encouragement

during the whole period. It is obvious that his attributive contribution and efforts have greatly

shaped me into what I am today. In fact, I am quite lucky to be a part of his ambitious

research team.

It is my great honor to convey my sincere gratitude to my respected teacher Professor Dr.

Md. Abdur Rashid, honorable Head of the Department of Chemistry, BUET for giving me

his wonderful support to move through the academic processes during this M.Phil. program. I

would like to convey my deepest gratitude to Professor, Dr. Md. Nazrul Islam, Dr. Shakila

Rahman, Dr. Md. Shakhawat Hossain Firoz, Dr. Ayesha Sharmin and Dina Nasrin,

Department of Chemistry, BUET, for their valuable suggestions, appreciated comments,

guidance and help during the research period.

I am thankful to all other respected teachers of the Department of Chemistry, BUET, for their

time to time support. I would also like to thank all of the officers and staffs of the Department

of Chemistry, BUET for their continuous help during my study period.

I am highly thankful to Professor Dr. Hongbo Zeng, University of Alberta, Canada, for the

characterization of our samples using X-ray Photoelectron Spectroscopy (XPS) during my

research.

I am thankful to Nikhil Chandra Bhoumik, Md. Khairul Islam and Md. Emdad Hossain of

Wazed Miah Science Research Centre, Jahangirnagar University for their support and

suggestions.

I am highly grateful to all members of the board of examiners for their valuable suggestions

and appreciated comments.

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I am thankful to my dear colleagues and all the members of Azam Research Group for their

friendly cooperation and lovely encouragement throughout my research period.

I am also thankful to other fellows of Chemistry Materials Laboratory for their cooperation

during the research period.

I am grateful to the authority of BUET and The World Academy of Sciences (TWAS) for

providing financial support for this research work.

Finally, I would like to express my heartfelt indebtedness and profound gratitude to my

beloved father, mother and all of my family members for their continuous inspiration and

immeasurable sacrifices throughout the period of my study.

September, 2018 Nahida Akter

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Abstract Quantum dots (QDs) are tiny particles or nanocrystals of a semiconducting material in the

size range of 2-10 nanometers. QDs have size dependent emission wavelength tunability and

show unique luminescence properties, which can be exploited to use them in ion and

molecular detection. The luminescence method is one of the most promising approaches for

ion and molecular recognition owing to its high sensitivity. The luminescence or fluorescence

of the QDs gets quenched when they interact with other molecules or ions. The extent of this

quenching depends linearly on the concentration of the analytes. For this reason QDs are

being popular as fluorescent tags for the detection of ions or molecules involved in many

biological and biomedical fields. QDs have been successfully employed for the detection of

heavy metal ions, formaldehyde, dopamine, glucose, pesticides etc. In comparison to other

QDs, silicon quantum dots (SiQDs) have attracted much interest due to wide availability of

low cost, inert source materials, favorable biocompatibility, low cytotoxiciy and unique

optical and electronic properties. However, the selective detection of Hg2+ and melamine at

sub-ppm level using SiQDs has not been well explored yet. Herein, we exploited the specific

interaction strategy of Hg2+ and melamine with thymine and carried out thymine modification

of SiQDs. The presence of melamine or Hg2+ caused very rapid and significant fluorescence

quenching of SiQDs-Thy leading to the qualitative detection. From the fluorescence intensity

ratio change quantitative analysis was also possible and the limit of detection was as low as

0.005 µM for both Hg2+ and melamine. Such lower limit detection of Hg2+ and melamine

with QDs has not been reported yet. With increasing the concentration of Hg2+ or melamine,

the fluorescence intensity decreased gradually and such response was observed over a wide

concentration range. Moreover, SiQDs-Thy showed noticeable selectivity towards Hg2+ and

melamine compared to other ions and biomolecules. SiQDs-Thy thus show great promise as a

potential sensing platform for Hg2+ and melamine.

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Chapter 1 Introduction............................................................................................................ 1

1.1 Quantum dots ...................................................................................................................... 2

1.1.1 Principles of fluorescence…………………………………………………………..3

1.2 Some popular quantum dots….…...……………………………………………………….4

1.2.1 Carbon dots.…………………………………………………………........................5

1.2.2 Graphene quantum dot.…………………………………………………………….6

1.2.3 Silicon quantum dot..……………………………………………………………….7

1.2.4 CdSe and CdS quantum dots…………………………………………………..……8

1.3 Synthesis of quantum dots………………………………………………………………..11

1.3.1 Colloidal synthesis……………………………………………...…………………12

1.3.2 Plasma synthesis...…..……………………………………………………..………13

1.3.3 Lithographicassembly…...………..………………….…………………………….13

1.4 Application of quantum dots……………………………………………………………14

1.4.1 Bioimaging and biosensing………………………………………………………..14

1.4.2 Drug delivery………………………………………………………………………15

1.4.3 Quantum-computing.…………………...…...………………….………………….17

1.4.4 Solar cells………………………………………………………………………….17

1.4.5 Detection of ions and biomolecules…………………………………………………...18

1.4.5.1 Detection of Hg2+……………………………………………………………….20

1.4.5.2 Detection of melamine………………………………………………………….21

1.5 Motivation of our research work…………………………………………………………23

References……………………………………………………………………………………26

Chapter 2 Experimental ........................................................................................................ 37

2.1 Materials and instruments .................................................................................................. 38

2.1.1 Chemicals and reagents............................................................................................ 38

2.1.2 Instruments………………..……………………………………………………….39

2.2. Methods of preparation………………………………………………………………….39

2.2.1 Synthesis of silicon quantum dots…………………………………………………39

2.2.2 Thymine functionalization of silicon quantum dots……………………………….40

2.3 Detection of Hg2+ and melamine with SiQDs-Thy ………………………………..…….40

2.3.1 Sensitivity study of SiQDs-Thy towards Hg2+ and melamine ………………….40

2.3.2 Selectivity study of SiQDs-Thy towards Hg2+ and melamine…………………….41

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2.3.3 Kinetic study………………………………………………………………………41

2.4 Sample characterization………………………………………………………………….41

2.4.1 Field emission scanning electron microscopy (FESEM) analysis…………….….41

2.4.2 Ultraviolet-visible (UV-vis) analysis.…………….……………………………....42

2.4.4 Energy dispersive X-ray (EDX) analysis …………………………………...……42

2.4.4 X-ray Photoelectron Spectroscopy (XPS)………………………………………..42

2.4.5 Fourier transform infrared (FTIR) analysis……………………………………….42

2.4.6 Fluorescence spectroscopy analysis ………………….…………………………43

References……………………………………………………………………………………43

Chapter 3 Result and Discussion…………………………………………………………..44

3.1 Synthesis of thymine functionalized silicon quantum dots………………………............45

3.2 Field emission scanning electron microscopy (FESEM) analysis…………………….…47

3.3 UV-vis analysis of SiQDs and SiQDs-Thy ………………………………………….…..48

3.4 Energy dispersive X-ray (EDX) analysis………………………..…………………...…. 49

3.5 Surface composition analysis by X-ray Photoelectron Spectroscopy (XPS)………….....50

3.5.1 XPS survey spectrum analysis……………………………………………………50

3.5.2 High resolution C 1s spectrum analysis…………………………………….…….51

3.5.3 High resolution N 1s spectrum analysis…………………………...………….......52

3.6 Functional group analysis by FTIR…………….………………………………………...53

3.7 Fluorescence spectroscopy analysis……………………….………………….………….54

3.8 Detection of Hg2+ with SiQDs-Thy…………………………………………….………...56 3.8.1 Sensitivity of SiQDs-Thy toward Hg2+…………………………………………...56

3.8.2 Kinetic study………………………………………………………………………58

3.7 Detection of melamine with SiQDs-Thy ………………………………………………...58

3.9.1 Sensitivity of SiQDs-Thy toward melamine……………………………………...58

3.9.2 Kinetic study……………………………………………………………….……...59

3.10 Selectivity of SiQDs-Thy towards Hg2+ and melamine………………..……………….60

3.11 Conclusion………………………………………..……………..…………………..…..61

References……………………………………………………………………………………62

CHAPTER 1

Introduction

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1.1 Quantum Dots

Quantum dots (QDs) are very tiny particles or nanocrystals of a semiconducting material with

diameters in the range of 2-10 nanometers (10-50 atoms). QDs were first discovered by Alexey

Ekimov in 1980 [1]. Now-a-days, nanoscale materials are of huge research interest due to their

efficiency in developing new methods, new devices, new tools, and to investigate different

application fields. Especially, a new generation of highly efficient technology is being developed

employing QDs as building block material [2].

Due to very small size, the electrons in QDs are confined in a small space leading to quantum

confinement effect [3, 4]. The discrete, quantized energy levels of QDs are similar that of atoms

and for this reason QDs are also known as 'artificial atoms'.

Figure 1.1. Quantum confinement effect in quantum dots

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With decrease in the size of QDs, the energy difference between the highest valence band and

the lowest conduction band increases. More energy is then required to excite the dot and more

energy is released when the QD returns to its ground state. As a result, QDs can emit any color

of light from the same material simply by changing the dot size. Larger QDs have smaller energy

band gaps yielding a red shifting of their absorption bands and the emitted red light. Conversely,

smaller QDs have a larger energy band gap resulting in a blue shifting and the emission of blue

light. Because of the high level of control possible over the size of the nanocrystals produced,

QDs can be tuned during manufacturing to emit any color of light [5].

1.1.1 Fluorescence properties of QDs

Quantum dots display unique electronic properties, intermediate between those of bulk

semiconductors and discrete molecules [6, 7]. The most apparent result of this is fluorescence

[8]. Also compared with the traditional organic fluorophores (e.g., organic dyes and fluorescent

proteins), QDs have larger absorption coefficients, size-tunable light emission, superior signal

brightness, resistance to photobleaching and simultaneous excitation of multiple fluorescence

colors [9].

Fluorescence is the type of photoluminescence (PL) most commonly used in analytical chemistry

nowadays. First observed in 1565, its name originates from the mineral fluorspar, which shines

under ultraviolet (UV) radiation [10]. A major event in the history of photoluminescence was the

publication by Sir George Gabriel Stokes, physicist and professor of mathematics at Cambridge,

of his famous paper entitled ―On the Refrangibility of Light‖ in 1852 [11]. He clearly identified a

common phenomenon he called dispersive reflection: the wavelengths of the dispersed light are

always longer than the wavelength of the original light.

One of Stokes’s experiments that is spectacular and remarkable by its simplicity deserves

attention. Stokes formed the solar spectrum by means of a prism. When he moved a test tube

filled with a solution of quinine through the visible part of the spectrum, nothing happened: the

solution remained transparent [12]. But beyond the violet portion of the spectrum, that is, in the

invisible zone corresponding to ultraviolet radiation, the solution glowed with a blue light

(Figure 1.2). Stokes wrote [11], It was certainly a curious sight to see the tube instantaneously

light up when plunged into the invisible rays: it was literally darkness visible. Altogether the

phenomenon had something of an unearthly appearance. From his experiments with a wide

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range of substances, Stokes concluded that the dispersed light was always of longer wavelengths

than the incident light. Later this statement became the Stokes law.

Figure 1.2. Principle of Stokes’s experiment showing that a solution of quinine irradiated with

UV emits blue light, whereas no effect is observed when it is placed in the visible part of the

solar spectrum [12].

Stokes also noted that the dispersion of light took place in all directions, hence, the fluid behaved

as if it were self-luminous. In his paper, Stokes called the observed phenomenon true internal

dispersion or dispersive reflection but in a footnote [11], he wrote, I confess I do not like this

term. I am almost inclined to coin a word, and call the appearance fluorescence, from fluorspar,

as the analogous term opalescence is derived from the name of a mineral.

Fluorescence differs from other photoluminescence phenomena because the excited molecule

remains in the excited state for a very short time (on the order of 10 − 9 s). At room temperature

thermal energy is not adequate to significantly populate the excited vibrational states due to

larger energy difference between ground and excited states. For this reason light energy is used

instead of heat to induce fluorescence. The processes that take place during light absorption and

emission could be illustrated by the renowned Jablonski diagram. A schematic diagram is as

shown in Figure 1.3.

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Figure 1.3. A brief illustration of the Jablonski diagram

When a molecule absorbs light, an electron is promoted to a higher excited state (generally a

singlet state). Singlet ground, first and second electronic states are represented by S0, S1 and S2

respectively with vibrational energy levels depicted by 0, 1 and 2 existed in fluorophores. Triplet

states are represented by T1, T2 etc. Transitions between states are illustrated by vertical lines.

The excited state can get depopulated in several ways.

The molecule can lose its energy non–radiatively by giving its energy to another

absorbing species in its immediate vicinity (energy transfer) or by collisions with other

species in the medium.

If an excited state triplet overlaps with the exited state singlet, the molecule can cross

over into this triplet state. This is known as inter system crossing. If the molecule then

returns to the ground state singlet (T1→S0) by emitting light, the process is known as

phosphorescence.

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The molecule can partially dissipate its energy by undergoing conformational changes

and relaxed to the lowest vibrational level of the excited state in a process called

vibrational relaxation. If the molecule is rigid and cannot vibrationally relax to the ground

state, it then returns to the ground state (S1→S0) by emitting light, the process is known

as fluorescence.

From the Jablonski diagram, it was clear that energy of emission is less than that of absorption.

This is reflected by fluorescence which occurs at longer wavelengths or lower energies, clearly

indicating energy losses between excitation and emission. This occurrence was perceived by Sir.

George G. Stokes in 1852 at University of Cambridge (Stokes, 1852). Stokes shift is the

difference (in wavelength or frequency units) between positions of the band maxima of

the absorption and emission spectra of the same electronic transition. One usual reason of Stokes

shift is the rapid energy deterioration of fluorophore to the lowest vibrational level of S1.

Moreover, solvent effects, complex formation and energy transfer can cause further Stokes

shifts.

Figure 1.4. Absorption and emission spectra of Rhodamine 6G with ⁓25 nm Stokes shift [13].

A fluorophore is a fluorescent chemical compound that can re-emit light upon light

excitation. The fluorophore absorbs light energy of a specific wavelength and re-emits light at a

longer wavelength. Fluorophores typically contain several combined aromatic groups, or planar

or cyclic molecules with several π bonds (Figure 1.5).

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Figure 1.5. Some common fluorophores

The fluorescence lifetime and quantum yield are perhaps the most important characteristics of a

fluorophore. Quantum yield is the number of emitted photons relative to the number of absorbed

photons. Substances with the largest quantum yields, approaching unity, such as rhodamines

(0.78 in alcohol), display the brightest emissions. The lifetime is also important, as it determines

the time available for the fluorophore to interact with or diffuse in its environment, and hence the

information available from its emission. Fluorescence lifetime and quantum yield are best

represented by a simplified Jablonski diagram (Figure 1.6).

Figure 1.6. A simplified Jablonski diagram to illustrate the meaning of quantum yield and

lifetime

The fluorescence quantum yield is the ratio of the number of photons emitted to the number

absorbed. The rate constants Γ and knr both depopulate the excited state. The fraction of

fluorophores that decay through emission, and hence the quantum yield, is given by

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The lifetime of the excited state is defined by the average time the molecule spends in the excited

state prior to return to the ground state. Generally, fluorescence lifetimes are near 10 ns. The

lifetime of a fluorophore is given by

Γ

1.2 Some popular quantum dots

1.2.1 Carbon dots

Carbon dots (CDs), as a promising new class of fluorescent nanoparticles, have recently received

immense research interest. CDs generally refer to small carbon nanoparticles with sizes below 10

nm. Oxygen, hydrogen and other elements may also exist in the small dots [14]. Compared with

conventional semiconductor QDs, CDs exhibit many unique merits such as chemical inertness

and stability, high photostability against photobleaching, tunable excitation (Figure 1.7) and

emission spectra, excellent biocompatibility and low toxicity [15].

Figure 1.7. Aqueous solutions of the carbon dots excited at the indicated wavelengths in nm [16]

There have been many functional groups and/or passivation agents used to cover the surface of

the CDs outside the carbon core, endowing CDs with high quantum yield (QY), chemical

stability and good water solubility. In addition, starting materials for CDs are abundantly

available, resulting in the possibility of low cost and mass production of the CDs [14]. CDs have

been demonstrated for successful uses in the fuorescence imaging of cells [17], taking advantage

of their visible excitation and emission wavelengths, their fuorescence brightness at the

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individual dot level, and their high photostability. CDs also act as effective visible-light

photocatalysts for oxidation and reduction reactions [18]. The same photoinduced redox

processes responsible for the photocatalytic activities make CDs an excellent candidate as

antibacterial agents [19]. CDs also have wide range of applications (Figure 1.8) in biomedicine

as drug carriers, fluorescent tracers as well as controlled drug release [20]. CDs are used as

photosensitizers in photodynamic therapy to destroy cancer cells [21].

Figure 1.8. Important applications of GQDs and C-dots

1.2.2 Graphene quantum dots

Fluorescent graphene quantum dots (GQDs) have attracted tremendous attention because of their

unique 2D layered structure, with lateral dimensions less than 100 nm in single, double or few

(3–10) layers [22]. Photoluminescence (PL) of GQDs can be easily tailored by controlling their

size and shape, doping nonmetallic ions, and modifying their surfaces and edges [22]. GQDs also

exhibit large surface area, ease of surface modification, high photostability, low cytotoxicity, and

excellent biocompatibility [23].

Various novel methods have been developed to prepare high quality, fluorescent GQDs.

Generally, the syntheses of GQDs can be divided into two main categories, top-down and

bottom-up synthetic approaches, based on the relationship between the source and product [22,

24]. Top down strategies involve directly cleaving bulk carbon materials into nanoscale GQDs

via physical, chemical or electrochemical techniques [25]. Conversely, bottom-up methods are

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growth strategies; namely, appropriate molecular precursors gradually grow into nanosized

GQDs by applying external energy. GQDs have been widely used for a variety of purposes,

including production of photovoltaic devices [26], organic light-emitting diodes [27], fuel cells

[28] and drug-delivery systems [29]. GQD-based fluorescent sensors show promise in detection

of inorganic ions [30], small organic molecules [31] and large biomaterials [32]. In recent years,

they also found many applications in analytical chemistry (Figure 1.8) in response to the

increasing research interest aroused by their unique properties. Thus, GQDs have emerged as

new potential tools for designing and tuning PL sensors and biosensors.

1.2.3 Silicon quantum dots

Silicon quantum dots (SiQDs) hold great promise for many future technologies. Recently,

silicon-based nanostructures have drawn extensive attention, due to the wide availability of the

source materials to make such nanostructures, and the superb electronic, optical, catalytical, and

mechanical properties of these silicon nanomaterials [33, 34]. With respect to bulk silicon, the

optical and electronic properties of SiQDs are dramatically modified by and dependent on the

QD size. SiQDs have been widely used in biology, owing to their good biocompatibility, low

cytotoxicity, and antiphotobleaching capability [35, 36]. As excellent fluorescent probes, SiQDs

can be used in ion detection [37] and long-term bioimaging/biosensing [38]. It has been

demonstrated that (Figure 1.9) SiQDs are capable of producing bright and spectrally tunable

emission [39].

(a) (b) (c)

Figure 1.9. (a) SiQDs with visible emission[40] (b) SiQDs with UV emission [39, 41] (c)

NIR/red emitting SiQDs [42, 43]

The common methods of preparing Si QDs can be classified into two types: two-step synthesis

and one-step synthesis. The two-step synthesis procedure includes liquid reduction method [44],

photochemical etching method [45], high temperature pyrolysis method [46] and plasma

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synthesis [47]. The universally -used silicon sources in two-step synthesis methods are silica

powder, silicon halides, silicon oxides etc. [48] leading to poor water solubility of prepared Si

QDs and inferior stability caused by easy oxidation tendency. On the other hand, the one-step

synthesis methods contains microwave method [49], ultraviolet radiation method [50] and

hydrothermal route [51]. Hydrothermal route requires no complicated and plentiful equipment,

and is environmental friendly and convenient in operation, making it an appropriate method for

Si QDs preparation. However, most of the fluorescence emissions of Si QDs prepared by

hydrothermal route are blue, which can be easily interfered by tissue scatter and auto-

fluorescence.

1.2.4 CdSe and CdS quantum dots:

CdSe quantum dots (CdSe QDs) are widely investigated as their emissions can be easily tuned

from red to blue with decreasing nanoparticle size [52]. As II-VI semiconductor nanoparticles,

CdSe QDs exhibit strong confinement of excited electrons and holes, which leads to dramatically

different optical and electronic properties compared to bulk CdSe [53]. They also possess high

luminescence quantum yield and narrow band gap [54]. CdSe QDs find a wide range of

applications in optoelectronic devices [55], photo catalysis, solar energy conversion and

biological imaging and labeling.

CdS QDs have also been extensively studied due to their potential applications in several

technological areas such as solar photovoltaic cells, nano bar codes, field effect transistors, light

emitting diodes, photocatalysis and in vivo biomedical detection fluorescent tags in biology and

the development of chemical and biological sensors [56, 57]. CdS QDs have been identified as a

potential candidate to diagnose cancerous cells [58]. Water-soluble luminescent CdS quantum

dots (QDs) capped by polyphosphate, L-cysteine, and thioglycerol can detect physiologically

important metal cations [59].

1.3 Synthesis of quantum dots

Because of the great utility of quantum dots, many methods of synthesis have been proposed and

implemented. These vary greatly in terms of scalability, quality, and ease.

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1.3.1 Colloidal synthesis

The most accessible technique for creating quantum dots is colloidal synthesis - that is,

chemically producing quantum dots suspended in solution. In general, a saturated solution of the

semiconductor is produced in an organic solvent. Then the temperature or pH is changed to

produce a supersaturated solution, which nucleates to produce small crystals. The size of the

resulting quantum dots can typically be tuned by changing the temperature, pH, or length of the

reactions. Most of these processes can be carried out in small quantities in the lab without

requiring exotic reagents or equipment, although larger quantities can be difficult to produce as

precise temperature control is required.

For the colloidal synthesis of CdSe (Figure 1.10), a cadmium compound is heated to 320 °C and

dissolves in an organic solvent [60]. A room temperature selenium compound dissolved in a

different organic solvent is injected into the reaction vessel, causing supersaturation of the

resultant CdSe solution. As the temperature drops to around 290 °C, nucleation of new crystals

stops and existing crystals grow.

Figure 1.10. Colloidal synthesis of CdSe [60]

After a period of growth the solution is cooled to 220 °C stopping growth. A small amount of

zinc sulfide is injected into the reaction vessel to coat the quantum dots and prevent them from

reacting with the environment [60].

1.3.2 Plasma synthesis

Plasma synthesis has evolved to be one of the most popular gas-phase approaches for the

production of quantum dots, especially those with covalent bonds [42]. For example, silicon (Si)

13

and germanium (Ge) quantum dots have been synthesized by using nonthermal plasma. The size,

shape, surface and composition of quantum dots can all be controlled in nonthermal plasma

[61]. Quantum dots synthesized by plasma are usually in the form of powder, for which surface

modification may be carried out. This can lead to excellent dispersion of quantum dots in either

organic solvents [62] or water [63].

1.3.3 Lithographic assembly

Semiconductor lithography is typically used for the construction of integrated circuits. A

semiconductor wafer is coated with a photosensitive ―resist‖ material, which is then covered with

a stencil and exposed to ultraviolet light, which causes the parts exposed to light to harden

(Figure 1.11). Photolithographic techniques are sometimes used for producing quantum dots

[64], but the small size (∼ 10 nm) desired in quantum dots can be hard to achieve with even

high-frequency ultraviolet light. Instead, a non-conventional form of lithography called imprint

lithography is best suited for producing quantum dots [65].

Figure 1.11. Lithographic assembly. A nanoscale mold is pressed into a semiconductor to create

very small features [65]

Instead of etching a semiconductor wafer with light, a negative image is formed on a hard SiO2

wafer using electron-beam lithography, a process similar to photolithography. This mold is used

as a stamp and is physically pressed onto a silicon layer to form the quantum dots. This

technique allows for precise positioning and size control of the quantum dots, and is being

investigated for use in classical and quantum computing applications.

14

1.4 Application of quantum dots 1.4.1 Bioimaging and biosensing

QDs are widely used as labels and contrast agents for in vitro and in vivo imaging. QDs offer

very appropriate sizes for cell labeling, unique optical and electrical properties [66], surface

functionalization and multimeric binding capacities [67, 68]. The size of labels is especially

important in cell analysis and imaging. The smallest probes (1-10 nm) are preferred since they do

not influence the biomolecule’s mobility or ability to interact with its environment. QDs thus

provide enhanced targeting efficiencies, in particular, for areas in which long-term PL stability,

high brightness or multi-colour detection are crucial [69]. QDs are also considerably brighter and

more resistant to photobleaching than are organic dyes and fluorescent proteins. These properties

are well suited for dynamic imaging at the single-molecule level and for multiplexed biomedical

diagnostics at ultrahigh sensitivity (Figure 1.12).

Figure 1.12. Representative fluorescence and phase-contrast images of different cell types

labeled with CdS CQDs bound to (A) neuroblastoma cells, (B) pheochromocytoma cells, and (C)

rat neonatal cortical cells [70]. Scale bar: 25 µm.

Gao et al. demonstrated the preparation of anti-body-conjugated polymeric micelle coated

CdSe/ZnS QDs for targeting and imaging of human prostate cancer growing in nude mice [71].

Cai et al. reported the in vivo targeting and imaging of tumor vascula-ture using arginine-

glycine-aspartic acid (RGD) pep-tide-labeled CdTe/ZnS quantum dots (QDs) [72].

15

Developments in QD surface engineering have enabled them to be used as novel fluorescent

probes for sensing and analyzing a wide variety of chemical and biological analysts. For

example, competitive fluoroimmunoassay based on QD antibody conjugates has been developed

for detection of different targets ranging from small molecules to proteins, virus and bacteria

[73]. In addition to the analyzing abilities of QDs, recent advances in QD-based immunoassay

with ultrahigh sensitivity or rapid screening capability have also benefited the developments of

novel readout methods, such as electrochemical detection, barcode, microfluidics technique and

other readout strategies [74]. QDs are considered as spectacular FRET donor candidates for bio-

sensing applications. FRET sensing using QD as the donor has been extensively reported for

DNA/RNA sequence monitoring and non-genetic molecule detection [75].

1.4.2 Drug delivery

Drug delivery is a field of vital importance to medicine and healthcare. Controlled drug delivery

to targeted cells or tissues can improve bioavailability by preventing premature degradation,

enhancing uptake and reduce side effects by targeting only the disease site or target cells [76].

QDs have been engineered to carry different classes of therapeutic agents for simultaneous

imaging and therapeutic applications. The formulation of QD drug conjugates would facilitate

the elucidation of the mechanistic pathways of drug delivery as well as the diagnosis and

treatment of many diseases. Manabe et al. conjugated QDs to captopril (QD-cap). By measuring

the fluorescence intensity of the QDs, they analyzed the QDcap concentration in the blood and

the small-molecule dynamics and kinetics, which revealed that QD-caps had approximately the

same half-life as captopril. In addition, the QD fluorescence revealed that the QD-caps

accumulated in the liver, lungs, and spleen [77].

Chakravarthy et al. reported on the ability of nanoconjugates of CdSe/CdS/ZnS QD and doxoru-

bicin to target alveolar macrophages, cells that play a critical role in the pathogenesis of

inflammatory lung injuries [78]. Confocal imaging showed the release of Dox from the QD-Dox

nanoconjugate, confirmed by its accumulation in the cell nucleus and induction of apoptosis,

indicating that the drug retains its bio-activity after coupling to the nanoparticle (Figure 1.13).

16

Figure 1.13. Schematic illustration of (a) QD−Apt(Dox) Bi-FRET system. (b) specific uptake of

QD−Apt(Dox) conjugates into target cancer cell through PSMA mediated endocytosis. The

release of Dox from the QD−Apt(Dox) conjugates induces the recovery of fluorescence from

both QD and Dox, thereby sensing the intracellular delivery of Dox and enabling the

synchronous fluorescent localization and killing of cancer cells [79].

1.4.3 Quantum computing

Quantum dot technology is one of the most promising candidates for use in solid-state quantum

computation. By applying small voltages to the leads, the flow of electrons through the quantum

dot can be controlled and thereby precise measurements of the spin and other properties therein

can be made. Two nearby quantum dots, each with a single excited electron, have been used as a

two-qubit system for quantum computation [80]. The system is placed in a uniform magnetic

field, creating a splitting between the | ↓› and | ↑› electron states. The system can be initialized in

the | ↑↓i state by putting both electrons in a single well, which has a singlet state as the ground

state. As the potential of the second well is lowered adiabatically, one of the electrons will move

into that well, depending on the polarity of the magnetic field. Allowing a coupling between the

two electrons then allows a √ SW AP gate to be applied, which exchanges the | ↑↓› and | ↓› states

17

when applied twice. This gate, when paired with single qubit operations, is known to be

universal – that is, it can be used to create any quantum circuit [81].

1.4.4 Solar cells

Extensive research has been carried out by a significant number of researchers in the field of

solar cells, which convert the sunlight, the most interesting renewable energy resource, to

electricity. Among next-generation photovoltaic systems QD-based solar cells stand out as a very

promising candidate due to unique and versatile advantages of QDs, such as wide tunability of

band gaps, easy solution processability, impressive capability of generating multiple excitons

and slowing down the cooling of hot electrons. Five important classes of QD-based solar cell

devices are QD-sensitized solar cell, hybrid QD-polymer solar cell, Schottky junction solar cell,

p-n heterojunction solar cell, and p-n homojunction solar cell. Almost all of these solar cells

favor the use of presynthesized monodisperse, high-quality QDs to avoid possible charge carrier

trapping and recombination in relatively larger QDs. The cells are typically made up by coupling

presynthesized semiconductor QDs to other widerband- gap semiconductor materials, mainly

TiO2 (or ZnO) with favorable band offset, referred as a type-II structure, to enable the charge

separation at their interface.

QD-sensitized solar cells represent one of the earliest configurations of QD solar cells. The cells

are typically made up by coupling presynthesized semiconductor QDs to other widerband- gap

semiconductor materials, mainly TiO2 (or ZnO) with favorable band offset. The first Schottky

QD solar cell involving a thin and compact QD film was reported by Nozik’s group in 2008 [82].

This type of solar cells was once leading in the efficiency in all types of QD solar cells, with the

highest reported efficiency reaching 4.5% in 2011 [49]. The structure is quite simple and is

constructed by sandwiching the QD layer in between an Ohmic-contact transparent electrode

such as indium-doped tin oxide (ITO) and a low-work-function metal electrode [49]. In general,

electron-hole pairs are photogenerated in the active QD film and separated at the Schottky

junction imposed in between the back metallic contact and the semiconducting QD film. In

hybrid QD-polymer solar cells the single photoactive layer is prepared via blending QDs with

conjugated polymers, or alternatively QDs and polymers are arranged into separate layers to

build a bilayer or planar configuration cell [83]. The charge separation takes place at the

QD/polymer interface and is dictated by their energy levels. Charge separation at the

18

QD/semiconductor junction area is one of the key parameters for p-n heterojunction solar cells.

In this case, the charge separation will be influenced by the electronic structure of QDs, which,

once again, is dominated by the size of QDs. The optimal size and thereby the optimal band gap

will be determined by the interplay of multiple relevant factors. Sargent’s group studied the

photovoltaic performance of p-n heterojunction solar cells based on the PbS QDs/TiO2 junction

involving QDs of three different diameters and band gaps (3.7 nm: 1.3 eV; 4.3 nm: 1.1 eV; 5.5

nm: 0.9 eV) and found that 1.3 eV was the optimal band gap, yielding the largest open-circuit

voltage (VOC), short-circuit current density (ISC), fill factor (FF), and PCE. The p-n

homojunction QD solar cells represent the most recent development [73]. Their development

was largely enabled by the most recent advent on solid-state ligand exchange and on

understanding ligand effects on QD properties.

1.4.5 Detection of ions and biomolecules

Sensing of ions and biomolecules using QDs is a very active research field now-a-days. In

general, the interaction of ions or biomolecules with QDs induces a fluorescence quenching or

Fröster resonance energy transfer (FRET). Fluorescence quenching refers to any process that

decreases the fluorescence intensity of a sample. A variety of molecular interactions can result in

quenching (Figure 1.14). Collisional quenching occurs when the excited-state fluorophore is

deactivated upon contact with some other molecule in solution, which is called the quencher. In

this case the fluorophore is returned to the ground state during a diffusive encounter with the

quencher. The molecules are not chemically altered. For collisional fquenching the decrease in

intensity is described by the well-known stern-Volmer equation: [ ] [ ]

In this expression K is the Stern-Volmer quenching constant, kq is the bimolecular quenching

constant, is the unquenched lifetime, and [Q] is the quencher concentration. The Stern-Volmer

quenching constat K indicates the sensitivity of the fluorophore to a quencher. A wide variety of

molecules can act as collisional quenchers such as molecular oxygen, halogens i.e., Br, I or other

heavy atoms, amines and electron deficient molecules like acrylamide. Fluorophores can form

nonfluorescent complexes with quenchers. This process is referred to as static quenching since it

occurs in the ground state and does not rely on diffusion or molecular collisions.

19

Energy of an excited electron in a molecule can also be converted into heat rather than the

energy of a photon (photoemission). In some cases, such energy can be used to overcome a

reaction barrier and thus triggers decomposition of the molecule or bond dissociation without

photoemission. The excited electron can also be transferred to another molecule (inter-molecular

electron transfer) or to another part of the same molecule (intra-molecular electron transfer). The

latter case is likely to be occurred if the molecule has both electron donor and acceptor moieties.

Figure 1.14. Types of fluorescence quenching

Fluorescence resonance energy transfer (FRET) occurs whenever the emission spectrum of a

fluorophore or donor molecule overlaps with the absorption spectrum of an acceptor molecule.

The donor and acceptor are coupled by a dipole- dipole interaction. The extent of energy transfer

is determined by the distance between the donor and acceptor, and the extent of spectral overlap.

Fluorescence quenching has numerous applications in various detection processes. Song et al.,

reported that ClO- could selectively quench the fluorescence of SiQDs through the electron

transfer mechanism, thus SiQDs can be developed as a nanosensor for ClO- [84]. He and

coworkers reported that SiNPs showed a superb ability to selectively detect DA over other

20

common molecules in the cell, including amino acids, peptides, proteins, ions, and other

neurotransmitters. In this case the quenching effect of DA on SiNPs is caused by rster

resonance energy transfer (FRET) [85]. The sensitivity and selectivity of QDs toward specific

ions and biomoleclues can be enhanced significantly via surface functionalization of QDs.

Carboxylic carbon quantum dots (cCQDs) have been employed sufcessfully in the detection of

nucleic acid. The principle behind the sensing of nucleic acid lies in the different propensity of

single stranded DNA and double-stranded DNA to adsorb onto the surface of cCQD [86]. Highly

sensitive and selective detection of Fe3+ ion is possible using dopamine functionalized graphene

quantum dots (DA-GQDs). The reaction mechanism for Fe3+ detection is based on the

complexation and oxidation of dopamine. The selectivity of DA-GQDs sensing probe is

significantly excellent in the presence of other interfering metal ions [87].

1.4.5.1 Detection of Hg2+

Mercury is one of the environmental contaminants that have been paid great attention in recent

years. Emitted elementary mercury vapors from industrial and non-anthropogenic sources are

easily transported in the atmosphere across oceans and continents and eventually accumulate on

plants, in topsoil, and in waters as the ionized form (Hg2+) [88]. To date, the contamination of

drinking water by Hg2+ ions remains a big problem. Hg2+ exposure usually occurs from eating

large predatory fish and vegetables grown in contaminated soil, from the use of cosmetics.

Occupational exposure frequently occurs. Long-term exposure to high Hg2+ levels is deleterious,

causing permanent damage to the immune system, digestive system, central nervous system and

many other organs with serious sensory, motor, and cognitive disorders [89]. The World Health

Organization (WHO) and United States Environmental Protection Agency (U.S. EPA) have

regulated the maximum allowed levels of Hg2+ in drinking water to be 6 and 2 ppb, respectively.

Inorganic mercury can be transformed into the highly toxic form such as organic methylmercury

under the catalysis of microorganism. These organic mercury that can amass in human body

throughout the food web [89] and cause cinesipathy, disgnosia, cerebral lesion, impaired vision,

and even death [90]. For these reasons, the development of straightforward, highly sensitive and

selective methods for monitoring and detecting Hg2+ is of great importance.

21

Traditional methods for determining Hg2+ ions are flame atomic absorption spectrometry, anode-

stripping voltammetry, inductively coupled plasma mass spectrometry, and X-ray fluorescence

spectrometry [91]. Therefore, developing another sensitive, selective, and reliable analytical

technique for determining Hg2+ ions is necessary [92]. Although these could meet the demand of

sensitive and selective measurement of Hg2+ in water samples, all these methods are not only

time-consuming, labor-intensive, and laboratory-based but also require expensive

instrumentation, well-trained personnel and large sample volume. As a result, the detection

methods that meet with the criteria of facility, rapidness, and portability as well as accuracy are

especially in demand. Fluorescence is one of the most powerful optical techniques for detecting

trace concentrations of mercury. Yuan et al. reported the use of 2-hydroxyethyldithiocarbamate

(HDTC)-modified CdSe-ZnS QDs (Figure 1.15) for the highly sensitive and selective detection

of Hg2+ [93].

Figure 1.15. Fluorescence Quenching of HTDC-QDs by Hg2+ [93]

The fluorescence of the aqueous HDTC modified QDs (HDTC-QDs) could be selectively and

efficiently quenched by Hg2+ through a surface chelating reaction between HDTC and Hg2+. The

orange fluorescence of the HDTC-QDs gradually changes to red upon the increasing amount of

Hg2+ added besides the decreasing of the fluorescence intensity.

1.4.5.2 Detection of melamine

Melamine (1,3,5-triazine-2,4,6-triamine) is a basic organic compound, which has been widely

used as a raw material in the chemical industry. It is commonly used in the production of

22

plastics, glues, and laminates and as a fertilizer [94]. Because of its high nitrogen level (66%

nitrogen by mass), melamine shows the analytical characteristics of protein molecules when the

Kjeldahl method is used for protein analysis in food. Thus, melamine can be used to deliberately

boost the apparent protein content of food products.[95].

However, the exposure to melamine may be particularly dangerous to human health. Melamine

has low acute oral toxicity, but its high concentrations can induce renal pathology and even death

in babies and children [96]. Melamine can be hydrolyzed to cyanuric acid which in turn

associates with melamine to form an insoluble melamine-cyanurate complex leading to stone

formation in the urinary system and acute renal failure by obstruction [97]. Besides, it will also

cause a variety of renal toxic effects such as nephrolithiasis, chronic kidney inflammation and

bladder carcinoma, high blood pressure, dizziness etc. [98].

It has been reported that hundreds of pets died of renal failure in 2007 and there were an

estimated 300 000 victims and 50 000 babies that were hospitalized from the infant formula

containing melamine in 2008 [99]. Therefore, the World Health Organization (WHO) has

recommended the tolerable daily intake for melamine to be 0.2 mg/kg body weight per day,

while, the US Food and Drug Administration (FDA) has updated the maximum residue levels of

melamine in infant formula to be 1.0 mg/kg and 2.5 mg/kg for milk and other milk products,

respectively [100].

Several instrumental methods such as mass spectrometry, capillary electrophoresis, liquid

chromatography, electrochemical methods, immunoassays, and Raman spectrometry are used to

determine the presence of melamine [101, 102]. Although these methods are accurate and

precise, they also are expensive, are time consuming, require tedious sample preparation, and are

not suitable for rapid on-site analysis. Fluorescence spectroscopy is a relatively simple, low-cost,

and portable analytical technique. A number of works for melamine monitoring directly

employing QDs as fluorescent probes have been reported. The principle of this strategy is based

on the enhancement and decrease of the fluorescence intensity of QDs induced by the interaction

between QDs and target analytes. Zhang et al. has developed a simple and sensitive fluorescence

senor for melamine detection in milk using water-soluble thioglycolic acid (TGA)-CdTe QDs of

different sizes [103]. Melamine could quench the fluorescence of TGA-CdTe QDs induced by

the change of surface properties via hydrogen bonding between the amine groups of melamine

and the carboxyl groups of the TGA-CdTe QDs at pH 8.0. Later, Wang et al. have proved a

23

sensitive and rapid method for melamine determination based on the fluorescence enhancement

of TGA-CdS QDs [104]. At lower solution pH of 3.0, TGA-CdS QDs exhibited weaker

fluorescence intensity in the absence of melamine. However, in the presence of melamine mixed

with PBS before addition, the fluorescence intensity was enhanced in that the protonated TGA

on the surface of QDs was replaced by the amine group of melamine which can attach onto the

surface of QDs via N-Cd bond.

1.5 Motivation of our research work

Rapid, sensitive, and selective detection of ions and biomolecules is of great importance in

various fields, such as biomedical diagnosis, drug screening, food safety, environmental

protection etc. [105, 106]. Even though a number of sensors are commercially available, novel

kinds of sensors with ease of portability, sufficient sensitivity, high specificity, excellent

reproducibility, multiplexing detection capability, and low cost remain in high demand.

Fluorescent sensors with high sensitivity, short response time, easy portability, minimal

equipment requirements, and low cost have therefore attracted extensive attention [14, 107].

Among them, fluorescent colloidal II–VI QDs have become very popular due to their unique

optical merits, such as high photoluminescence quantum yield (PLQY), robust photostability,

and sharp and tunable emission spectra, thereby allowing prolonged and multiplexed detection

[108]. Silicon, as the second-most abundant element in earth’s crust guarantees a rich and low-

cost resource support for a variety of silicon-related applications, especially for the modern

semiconductor industry [109]. Compared to bulk silicon, nanometric-sized silicon shows

distinct photoluminescence that benefits from the quantum confinement effect when the

particle size is generally less than 5 nm [110]. In addition to the strong fluorescence, the

silicon nanomaterials usually feature photostability superior to that of common organic dyes

and most semiconductor nanomaterials.

SiQDs have a wide range of applications in the detection of ions and biomolecules [37, 111]. But

low level of sensitivity and selectivity often limit their applications. In order to improve their

performance, QDs may be functionalized with molecules and compounds possessing specific

affinities for target analytes leading to the specific and selective detection of analytes of interest.

In our research work, we redesigned the functional surfaces of photoluminescent SiQDs with

thymine (Thy). It is one of the four nitrogenous nucleobases that form the basic building blocks

24

of DNA. We found that there are amino groups on the surface of the SiQDs, which can be

exploited for thymine modification. Thymine was appended on the surface of SiQDs via amide

coupling reaction between SiQD and thymine-1-acetic acid to form thymine substituted SiQD

(SiQDs-Thy) [112]. Even though thymine functionalized graphene quantum dots (GQDs) [113]

carbon dots (CDs) have been reported [102], thymine functionalization of SiQDs has not been

performed yet. The synthesis of SiQDs-Thy was aimed at creating functional probe materials for

the selective and specific recognition of Hg2+ and melamine.

Figure 1.16. Interaction of thymine with Hg2+ and melamine

Thymine interacts with Hg2+ leading to Thy-Hg2+-Thy pairs, which quench the fluorescence

emission of T-SiQD [112, 114]. The quenching is then used for sensing by the fluorescence

―turn-O ‖ process. Thymine also possesses complementary NH---O and NH---N hydrogen

bonds with melamine, thus offers a good choice for melamine detection based on hydrogen

bond-based strategy [115]. Figure 1.17 shows schematic presentation of interaction between

SiQDs-Thy and Hg2+ or melamine. SiQDs-Thy under UV-light at 365 nm shows bright blue

luminescence. Usually SiQDs-Thy remain dispersed in the solution the presence of melamine or

Hg2+ leads to aggregation and the resulting interaction causes significant fluorescence quenching.

25

So qualitative detection will be easier and also from the fluorescence intensity ratio change the

quantitative analysis is also possible.

Figure 1.17. Schemetic presentation of SiQD-Thy based detection of melamine and Hg2+

26

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CHAPTER 2

Experimental

38

2.1 Materials and instruments

2.1.1 Chemicals and reagents

The chemicals and reagents used in this research were of analytical grade and used without

further purification. Deionized water was used as solvent to prepare most of the solutions of this

work. The chemicals and reagents which were used in this research are listed below:

i. Trisodium citrate dihydrate (Merck)

ii. (3‐ Aminopropyl)trimethoxysilane, APTMS (Sigma‐ Aldrich)

iii. N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, EDC

(Sigma‐ Aldrich)

iv. N-hydroxysuccinimide, NHS (Sigma‐ Aldrich)

v. Thymine-1-acetic acid (Sigma‐ Aldrich)

vi. Mercury (II) chloride, HgCl2 (Merck)

39

vii. Melamine (Sigma‐ Aldrich)

viii. Sodium chloride, NaCl (Merck)

ix. Potassium chloride, KCl (Merck)

x. Magnesium chloride (Merck)

xi. Calcium carbonate (Merck)

xii. Ferrous chloride, FeCl2 (Merck)

xiii. Dopamine hydrochloride (Sigma‐ Aldrich)

xiv. Glucose (Sigma‐ Aldrich)

xv. Disodium hydrogen phosphate. Na2HPO4, (Merck)

xvi. Potassium dihydrogen phosphate, KH2PO4, (Merck)

2.1.2 Instruments

Analysis of the samples was performed using the following instruments:

I. Fluorescence Spectrophotometer (F 7000 FL Spectrophotometer-5J14000)

II. UV-visible Spectrophotometer (Shimadzu-1800)

III. Fourier Transform Infrared Spectrophotometer (SHIMADZU FTIR-8400)

IV. Field Emission Scanning Electron Microscopy (JSM-7600F, Tokyo, Japan)

V. X-ray photoelectron Spectroscopy (XPS)

VI. Centrifuge machine (Hettich, Universal 16A)

VII. pH meter (Hanna, HI 8424, Romania)

VIII. Digital Balance (AB 265/S/SACT METTLER, Toleto, Switzerland)

IX. Freeze dryer (Heto FD3)

X. Oven (Lab Tech, LDO-030E)

2.2 Methods of preparation

2.2.1 Synthesis of silicon quantum dots

Amino-coated silicon quatum dots (SiQDs) were synthesized following one-pot hydrothermal

process [1, 2]. APTMS was used as the source of silicon and trisodium citrate dihydrate as the

reduction reagent. In a 50 mL round bottom flask, 0.551 g of trisodium citrate dihydrate

dissolved in 13.0 mL of water was bubbled with nitrogen gas for about 10 min to remove

40

oxygen. Then 3.0 mL of APTMS was added into the above solution under vigorous stirring. In

presence of nitrogen gas, the stirring was continued for further 20 min to form SiQD precursors.

The resultant solution was then transferred into a 50 mL Teflon-lined stainless steel autoclave.

The autoclave was sealed and maintained at 180 °C for 20 h [2]. When the reaction was

complete, the autoclave was naturally cooled to room temperature. The purification of SiQDs

was carried out using a dialysis tube (molecular cut-off: 1000 Da). The dialysis tube was washed

thoroughly with distilled water before using. Finally, the purified solution of SiQDs was

collected and stored at 4 °C for use.

2.2.2 Thymine functionalization of silicon quantum dots

The thymine (Thy) moieties were conjugated onto the surface of SiQDs via EDC/NHS coupling

chemistry [3, 4]. Amide bond was formed between the primary amine groups on the SiQDs and

the carboxyl groups of thymine-1-acetic acid (Thy-COOH). 0.08 g of Thy-COOH was added

into 3 mL of water followed by the addition of 0.04 g of EDC and 0.08 g of NHS. The solution

was stirred at room temperature for 1 h. Afterwards the SiQDs solution (5 mL) was added into

the above mixture. The reaction mixture was stirred at room temperature for 24 h. Finally, the

solution containing thymine functionalized SiQDs (SiQDs-Thy) was purified via dialysis (1000

Da, molecular weight cut-off) using distilled water. Dialysis was continued for 24 h at room

temperature and the purified solution of SiQDs-Thy was stored at 4 °C for further applications.

2.3 Detection of Hg2+ and melamine with SiQDs-Thy

2.3.1 Sensitivity study of SiQDs-Thy towards Hg2+ and melamine

Samples containing buffer solution (PBS buffer, 20 mM, pH 7.4), Hg2+ solution of different

concentrations and SiQDs-Thy solution (20 µg/mL) were made up to 5 mL [1, 5]. The emission

spectrum of the solution was then measured 40 min later. All optical measurements were

performed at room temperature under ambient conditions, and the excitation wavelength was 330

nm, slit width was set at 10 nm. The sensitivity of SiQDs-Thy towards melamine was tested

following the same procedure. The concentration of Hg2+ and melamine ranged from 0.005 to 13

µM.

41

2.3.2 Selectivity study of SiQDs-Thy towards Hg2+ and melamine

Since SiQDs-Thy are intended to be used for the detection of Hg2+ and melamine, the possible

fluorescence quenching effect of SiQDs-Thy caused by some other ions and biomolecules was

also studied. The fluorescence intensity of SiQDs-Thy exhibited a significant decrease after

adding Hg2+ and melamine to the solution, whereas other metal ions such as K+, Na+, Mg2+, Ca2+,

Fe2+ and biomolecules like dopamine, glucose did not cause significant fluorescence changes.

Samples were prepared containing buffer solution (PBS buffer, 20 mM, pH 7.4), ion/biomolecule

solution and SiQDs-Thy solution (20 µg/mL) with total volume up to 5 mL [1, 5]. The

concentration of the solutions of Hg2+ and melamine was 5 µM whereas the solutions of other

ions/biomolecules were prepared of 25 µM concentration.

2.3.3 Kinetic study

The effect of Hg2+ and melamine on the fluorescence intensity of SiQDs-Thy was also studied

with the course of time [6]. 20 µM Hg2+/ melamine solution was added to SiQDs-Thy solution

(20 µg/mL) in presence of PBS buffer (20 mM, pH 7.4). The change in fluorescence intensity

was studied upto 50 min.

2.4 Sample characterization

2.4.1 Field emission scanning electron microscopy (FESEM) analysis

The surface morphology of the synthesized SiQDs-Thy was studied using Field Emission

Scanning Electron Microscopy (FE-SEM). Samples were put on a silicon wafer. The silicon

wafer was first washed with distilled water well, then dipped into piranha solution for half an

hour and again was washed with distilled water and ethanol. The wafer was dried at 130 °C for

10 min and then samples were put on it. The wafer with sample was kept into an oven overnight

at 50 °C. The sample loaded strip was then mounted to a chamber that evacuated to ~ 10-3 to 10-4

torr and then a very thin gold layer (~few nanometers thick) were sputtered on the sample to

ensure the conductivity of the sample surface. The sample was then placed in the main SEM

chamber to view its surface. The microscope was operated at an accelerating voltage of 5.0 kV.

The system was computer interfaced and thus provides recording of the surface images in the

42

computer file for its use as hard copy. MIPAR software was used for processing FESEM images

and to extract the size distribution histogram.

2.4.2 Ultraviolet-visible (UV-vis) analysis

UV-vis absorption spectra were recorded at room temperature on a Shimadzu-1800 UV-vis

spectrometer equipped with 1cm quartz cell. The bandgap absorption of SiQDs and SiQDs-Thy

was obtained from the UV-vis spectra analysis. Distilled water was used as the reference.

2.4.3 Energy dispersive X-ray (EDX) analysis

EDX is an analytical technique used for the elemental analysis or chemical characterization of a

sample. It relies on an interaction of some source of X-ray excitation and a sample. Each element

has a unique atomic structure thus allowing a unique set of peaks on its electromagnetic emission

spectrum. EDX systems are actually attachments to Electron Microscopy instruments (Scanning

Electron Microscopy (SEM) or Transmission Electron Microscopy (TEM)) instruments where

the imaging capability of the microscope identifies the specimen of interest. We obtained our

EDX data along with the SEM analysis.

2.4.4 X-ray Photoelectron Spectroscopy (XPS) analysis

XPS measurements of samples were performed using the AXIS ULTRA spectrometer (Kratos

Analytical). The base pressure in the analytical chamber was <3 x10-8 Pa. The monochromatic

AlKα source (hν=1486.6 eV) was used at a power of 210 W. The photoelectron exit angle was

90⁰ , and the incident angle was 35.3⁰ from the plane of the surface. The analysis spot was 400

x 700 μm. The resolution of the instrument was 0.55 eV for Ag 3d peaks. Survey scans were

collected for binding energies from 1100 to 0 eV with analyzer pass energy of 160 eV and a step

of 0.35 eV. The high resolution spectra were run with a pass-energy of 20 eV and a step of 0.1

eV.

2.4.5 Fourier transform infrared (FTIR) analysis

The infrared spectra of SiQDs and SiQDs-Thy were recorded on a FTIR spectrometer in the

region of 4000 – 500 cm-1. The samples were dried well. A small volume of samples were taken

into vial and freeze dried to collect solid samples. The solid samples were then compressed in a

43

metal holder under a pressure of 8–10 tons to make a pellet. The pellet was then placed in the

path of IR beam for measurements.

2.4.6 Fluorescence spectroscopy analysis

Fluorescence measurements of samples were performed using the F 7000 FL Spectrophotometer-

5J14000. Stock solutions of the SiQDs-Thy (20 µg/mL) were prepared using distilled water. For

sensitivity study, solutions of Hg2+ and melamine were prepared of different concentrations

ranged from 0.005 to 13 µM. For selectivity study stock solutions of metal ions and

biomolecules were prepared of 25 µM concentration. All fluorescence spectra were recorded at

optimized detection conditions in the PBS buffer solution (20 mM, pH 7.4) at 25 °C.

References

[1] X. Xu, S. Ma, X. Xiao, Y. Hu, and D. Zhao, "The Preparation of High-Quality Water-

Soluble Silicon Quantum Dots and their Application in the Detection of Formaldehyde,"

RSC Adv., vol. 6, pp. 98899-98907, 2016.

[2] Y. Chen, Y. Bai, Z. Han, W. He, and Z. Guo, "Photoluminescence Imaging of Zn(2+) in

Living Systems," Chem. Soc. Rev., vol. 44, pp. 4517-46, 2015.

[3] Y. Li, J. Xu, and C. Sun, "Chemical Sensors and Biosensors for the Detection of

Melamine," RSC Adv., vol. 5, pp. 1125-1147, 2015.

[4] K. V. Chakravarthy, B. A. Davidson, J. D. Helinski, H. Ding, W. C. Law, K. T. Yong, et

al., "Doxorubicin-Conjugated Quantum Dots to Target Alveolar Macrophages and

Inflammation," Nanomedicine, vol. 7, pp. 88-96, 2011.

[5] H. Xu, S. Huang, C. Liao, Y. Li, B. Zheng, J. Du, et al., "Highly Selective and Sensitive

Fluorescence Probe Based on Thymine-Modified Carbon Dots for Hg2+ and L-Cysteine

Detection," RSC Adv., vol. 5, pp. 89121-89127, 2015.

[6] X. Zhang, X. Chen, S. Kai, H.-Y. Wang, J. Yang, F.-G. Wu, et al., "Highly Sensitive and

Selective Detection of Dopamine Using One-Pot Synthesized Highly Photoluminescent

Silicon Nanoparticles," Anal. Chem., vol. 87, pp. 3360-3365, 2015.

CHAPTER 3

Results and Discussion

45

3.1 Synthesis of thymine functionalized silicon quantum dots (SiQDs-Thy)

Silicon quantum dots (SiQDs) were first synthesized following a one-pot hydrothermal process

[1, 2]. Hydrothermal route is preferred over other methods i.e., microwave and ultraviolet

radiation methods since requires no complicated and plentiful equipment, environment friendly

and convenient in operation. Also, most of the fluorescence emissions of SiQDs prepared by

hydrothermal route are blue, which can be easily interfered by tissue scattering and auto-

fluorescence.

SiQD precursor solution was prepared at room temperature from APTMS and trisodiumcitrate

dihydrate. APTMS was used as the silicon source and trisodiumcitrate dihydrate as the reduction

reagent. The hydrothermal treatment of precursor solution was performed at 180 °C for 20 h [2].

Large silicon nanoparticles obtained from the hydrolysis of APTMS were divided into small

silicon nanoparticles with the assistance of and trisodiumcitrate dehydrate at high temperature.

These small silicon nanoparticles gradually grew into SiQDs with time. Any excess reagent

present in the SiQD solution was removed via dialysis and the purified SiQD solution was stored

at 4 °C for further use. When the aqueous solution of SiQDs was illuminated by handled UV

light at 365 nm, bright blue luminescence was observed. Since trisodium citrate dihydrate and

APTMS do not emit any fluorescence, thus the bright blue light was indeed the fluorescence

from the SiQDs.

Then thymine modification of SiQDs was carried out following EDC/NHS coupling chemistry

[3, 4]. Figure 3.1 represents the synthesis scheme for thymine functionalized SiQDs (SiQDs-

Thy). The reaction was carried out at room temperature for 24 h resulting in the formation of

amide bond between the primary amine groups on the SiQDs and the carboxyl groups of

thymine-1-acetic acid (Thy-COOH). The intermediate product of the EDC coupling is an

unstable ester, and can easily react with water to regenerate the carboxyl group. To prevent this,

NHS was reacted with the intermediate ester to produce a semistable amine reactive NHS-ester

[5]. The amine reactive NHS-ester then consistently reacted with the primary amine groups of

SiQDs leading to stable amide bond between SiQDS and Thy-COOH. The purification of

SiQDs-Thy was carried out using a dialysis tube (molecular weight cut-off:1000 Da) for about

24 h in dark and the water was changed after every 3h. Finally, the solution was collected and

stored at 4 °C for further analysis.

46

Figure 3.1. Reaction scheme for thymine functionalization of SiQDs

47

3.2 Field emission scanning electron microscopy (FESEM) analysis

To analyze the surface morphology and particle size of as prepared SiQDs-Thy, FESEM analysis

was performed. As observed in the FESEM images in Figure 3.12 (a) and (b), SiQDs-Thy were

uniformly dispersed without apparent aggregation. MIPAR image processing software was used

to process the FESEM image of SiQDs-Thy and to extract the particle size distributions. The

particle size distribution is shown along with the FESEM image in Figure 3.2 (c).

Figure 3.2. (a) FESEM image of SiQDs-Thy, (b) processed FESEM image of SiQDs-Thy and

(c) size distribution histogram after processing with MIPAR image analysis software

According to literature, the size of blue emitting SiQDs usually lies in the range of 2-3 nm [6, 7].

After thymine modification the increase in size of SiQD-Thy is obvious and the average sizes of

the prepared SiQDs-Thy were found to be 7.25 ± 1.0 nm. So the particles are still within the

required size range of QDs i.e., 2-10 nm. The presence of some particles of ⁓ 3 nm was also

observed which may be unmodified SiQDS. SiQDs-Thy showed excellent aqueous dispersibility

as like as SiQDs [8, 9] and the aqueous solution of SiQDs-Thy was highly transparent in the

ambient environment. But the solution showed bright blue luminescence under UV light at 365

48

nm indicating the retention of fluorescence property in SiQDs-Thy even after thymine

modification (Figure 3.3).

Figure 3.3. Aqueous solution of SiQDs-Thy under UV light at 365 nm (left) and day light (right)

3.3 UV-vis analysis of SiQDs and SiQDs-Thy

The UV-vis absorption spectrum of SiQDs (Figure 3.4) showed strong absorption at around 335

nm which matches with the previous reports [8, 9]. In the UV-vis spectrum of SiQDs-Thy, a

broad absorption band appeared in the region of 305 to 365 nm, which was ascribed to the n–p*

transition of the C=O and C=C of SiQDs-Thy. The strong absorption peak at around 272 nm

(Figure 3.4) proved the successful modification of thymine groups on the surface of SiQDs since

the similar peak was also observed in the spectrum of thymine-1-acetic acid (Thy-COOH) [10].

Figure 3.4. UV-vis absorption spectra of SiQDs and SiQDs-Thy

49

3.4 Energy dispersive X-ray (EDX) analysis

EDX analysis was performed to analyze the elemental composition of SiQDs-Thy. The EDX

pattern (Figure 3.5) revealed the presence of Si, C, O and N in SiQDs-Thy. Atom % of the

elements present in the SiQDs-Thy are also reflected in the EDX spectrum.

Figure 3.5. EDX spectrum of SiQDs-Thy

The percentages of atoms obtained from the EDX analysis (Table 3.1) was desirable according to

the chemical structure of SiQDs-Thy.

Table 3.1. Elemental composition obtained from the EDX analysis of SiQDs-Thy

Element Mass % Atom %

C 38.1 47.2

N 16.2 17.1

O 28.6 26.6

Si 17.1 9.1

50

3.5 Surface composition analysis by X-ray photoelectron spectroscopy (XPS)

3.5.1 XPS survey spectrum analysis

XPS is a surface sensitive quantitative spectroscopic technique. It helps in studying the surface

composition and analysis of the elements present. As shown in Figure 3.6, the four obvious

peaks at 284.6, 531.4, 101.8, 400.2 eV represent C1s, O1s, Si2p, N1s respectively, indicating the

presence of principle constituent elements of SiQDs-Thy.

Figure 3.6. XPS survey spectrum of SiQDs-Thy

Table 3.2. Percentages of the elements obtained from the XPS survey spectrum of SiQDs-Thy

Material Element percentages (%)

C N O Si

SiQDs-Thy 48.2 8.7 28.0 15.1

From the survey spectrum analysis, the presence of desirable percentages of N, O, Si in SiQDs-

Thy was confirmed. But the percentage of C was comparatively higher than expected which was

attributed to amino propyl groups on the surface of SiQDs remaining unmodified by thymine

moiety.

51

3.5.2 High resolution C 1s spectrum analysis

The high resolution C 1s XPS spectrum of SiQDs-Thy (Figure 3.7) can be deconvoluted into

three carbon states at 284.6 eV, 286.1 eV, and 288.4 eV which are attributed to C–C/C=C, C–

O/C–N and C=O, respectively. These bonds mainly come from the thymine moiety indicating

the thymine modification of SiQDs.

Figure 3.7. High resolution C1s spectrum of SiQDs-Thy

Table 3.3. Percentages of the bonds obtained from C 1s peak fitting for SiQDs-Thy

Material

Bond percentages (%)

C-C/C=C C-O/C-N C=O

SiQDs-Thy 52.46 33.6 13.9

52

3.5.3 High resolution N 1s spectrum analysis

However, the fitting analysis of the high resolution N 1s spectrum of SiQDs-Thy (Figure 3.8)

reveals two peaks at 399.8 and 400.9eV corresponding to C-N-C and N-H respectively. Thymine

moiety is the only source of these bonds in the product thus confirming thymine modification.

Figure 3.8. High resolution N1s spectrum of SiQDs-Thy

Table 3.4. Percentages of the functional groups obtained from N 1s peak fitting for SiQDs-Thy

Material Bond percentages (%)

C-N-C N-H

SiQDs-Thy 78.3 34.8

53

3.6 Functional group analysis by Fourier Transform Infrared Spectroscopy (FTIR)

FTIR is a common characterization tool to analyze the surface functional groups. FTIR spectra

of both SiQDs and SiQDs-Thy (Figure 3.9) were recorded and studied to ensure the surface

modification of SiQDs by thymine. These absorption peaks appeared in both SiQDs and SiQDs-

Thy: the band at 3600-3200 cm-1 was assigned to N-H stretching vibration; N-H bending

vibration appeared at 1582 cm-1; the bands located at around 2934 cm-1 and 2872 cm-1 were due

to C-H asymmetric and symmetric stretching vibrations respectively; the bands at 668 and

673cm-1 corresponded to Si-O-Si asymmetric stretching vibration of SiQDs-Thy and SiQDs

respectively. Si-O-Si symmetric stretching vibration appeared at around 1131 cm-1.

Figure 3.9. FTIR spectra of SiQDs and SiQDs-Thy

Some new peaks appeared in the FTIR spectrum of SiQDs-Thy. C=O stretching vibration gave

rise to a peak at around 1654 cm-1 which was due to amide bond formation after thymine

modification of SiQDs [1]. An intense, sharp peak at 1241 cm-1 corresponded to C-N stretching

vibration [11]. Thus the analysis of FTIR spectra revealed that thymine modification of SiQDs

was done successfully.

54

Table 3.5. Characteristic FTIR peaks and interpretations corresponding to SiQDS and SiQDS-Thy

Wavenumber (cm-1) Assignment

3500-3200 N-H stretching

2934 C-H asymmetric stretching

2872 C-H symmetric stretching

1654 C=O stretching

1582 N-H bending

1241 C-N stretching

668, 673 Si-O-Si asymmetric stretching

1131 Si-O-Si symmetric stretching

3.7 Fluorescence spectroscopy analysis

Fluorescence spectroscopy analysis of SiQDs and SiQDs-Thy was performed to confirm their

fluorescence behavior. Their excitation wavelength-dependent emission behavior were studied

first (Figure 3.10). With increasing the excitation wavelength from 340 nm to 370 nm a gradual

increase in fluorescence (FL) intensity was observed for SiQDs. Maximum FL emission was

observed at around 450 mm resulting from 370 nm excitation wavelength (Figure 3.10 a). Then

with further increasing the excitation wavelength, FL intensity started to decrease. SiQDs-Thy

also showed excitation wavelength dependent emission behavior. Maximum emission intensity

at 450 nm was observed when SiQDs-Thy were excited at 330 nm and FL intensity started to

decrease when the excitation wavelength was greater than 330 nm (Figure 3.10 b).

For both SiQDs and SiQDs-Thy maximum emission was observed at around 450 nm but at

different excitation wavelengths i.e., 370 nm and 330 nm respectively. 330 nm was selected as

the maximum excitation wavelength in the following experiments involving SiQDs-Thy. It was

55

found that the fluorescence intensity was decreased after thymine modification since all the

surface amino groups of SiQDs were not thymine functionalized.

Figure 3.10. Fluorescence emission spectra of SiQDs (a), SiQDs-Thy (b) at different excitation

wavelengths

To further analyze whether the optical absorption and fluorescence excitation of SiQDs-Thy take

place in the same region or not, the UV absorption spectrum was plotted along with the

fluorescence spectra (Figure 3.11). The optical absorption at around 335 nm matched with the

maximum fluorescence excitation and the corresponding fluorescence emission was observed at

around 450 nm.

56

Figure 3.11. UV-vis absorption (Abs), fluorescence excitation (Ex) and emission (Em) spectra

3.8 Detection of Hg2+ with SiQDs-Thy

3.8.1 Sensitivity study

The sensitivity of the detection system, SiQDs-Thy towards Hg2+ was studied via fluorescence

spectroscopy analysis. Figure 3.12 (a) shows the fluorescence emission response of SiQDs-Thy

interacting with various concentrations of Hg2+ ranging from 0.005 to 13 µM. The FL intensity at

450 nm decreased gradually with increasing the concentration of Hg2+. Figure 3.12 b shows the

relative change of the fluorescence intensity ratio (F/F0) at 330 nm as a function of

Hg2+concentration, [Hg2+] where F0 and F are the FL intensities in the absence and presence of

the quencher Hg2+, respectively. The plot of F/F0 at 330 nm versus [Hg2+] showed a linear

relationship with the square of correlation coefficient (R2) of 0.967231 (inset in Figure 3.12 b).

R2 is a statistical measure of how close the data are to the fitted regression line. Such an

outstanding FL quenching was assigned to the photoinduced electron transfer from the excitated

state of SiQDs-Thy to the vacant d orbital of Hg2+ when the T–Hg2+–T structure was formed [10,

12, 13]. The presented sensor (SiQDs-Thy) will thus allow the on-spot detection of Hg2+ in some

suspected highly contaminated sites in a prompt and specific way, in large contrast to those

depending on complicated instruments.

57

Figure 3.12. (a) Fluorescence intensity changes of SiQDs-Thy in the presence of different

concentration of Hg2+ (b) The plot of the fluorescence intensity ratio of SiQDs-Thy at 330 nm

versus the concentration of Hg2+

3.8.2 Kinetic study

The effect of time on the fluorescence intensity of SiQDs-Thy after adding 20.0 μM Hg2+ was

observed [7]. From the kinetic study it was found that the presence of mercury led to significant

fluorescence quenching immediately. After 45 min, the FL intensity of SiQDs-Thy decreased to

42.5% of initial intensity. The inset of Figure 3.13 represents SiQDs-Thy solution under

illumination of UV light at 330 nm, one in absence of Hg2+ solution (left) and another after 10

min of adding Hg2+ solution (right).

58

Figure 3.13. Fluorescence intensity change of SiQDs-Thy with time in presence of Hg2+. Inset:

photographs of SiQD-Thy (left) and SiQD-Thy in presence of Hg2+ (right) under illumination of

365 nm UV light

3.9 Detection of melamine using SiQDs-Thy

3.9.1 Sensitivity study

The effect of melamine concentration on the fluorescence intensity of SiQDs-Thy was also

studied via fluorescence spectroscopy analysis. Melamine solution of different concentrations

ranging from 0.005 to 10 µM was added and it was observed that the fluorescence intensity

decreased gradually with increasing concentration of mealmine (Figure 3.14). Melamine

interacts with thymine via H-bond formation [12] leading to significant fluorescence quenching

of SiQDs-Thy. Furthermore, the plot of quenching ratio (F/F0) vs. concentration of melamine,

[Melamine] showed linear response over a wide concentration range of melamine. Here, F0 and F

are the FL intensities in the absence and presence of the quencher melamine and the value of R2

is 0.997871. The sensitivity of SiQD-Thy towards melamine was proved from the successful

detection of only 0.005 µM of melamine in aqueous solution since lower limit detection for

melamine has not been reported yet using QDs.

59

Figure 3.14. (a) Fluorescence intensity changes of SiQDs-Thy in the presence of different

concentration of melamine (b) The plot of the fluorescence intensity ratio of SiQDs-Thy at 330

nm versus the concentration of melamine

3.9.2 Kinetic study

The change in fluorescence intensity of SiQDs-Thy with time in presence of melamine was

observed [7]. 20.0 μM melamine solution was added to SiQDs-Thy solution and after 50 min

fluorescence intensity decreased to 44.04% of the original intensity. The inset of Figure 3.15

represents SiQDs-Thy solution under illumination of UV light at 330 nm, one in absence of

melamine solution (left) and another after 30 min of adding melamine solution (right).

60

Figure 3.15. Fluorescence intensity change of SiQDs-Thy with time in presence of melamine.

Inset: photographs of SiQD-Thy (left) and SiQD-Thy in presence of melamine (right) under

illumination of 365 nm UV light

3.10 Selectivity of SiQDs-Thy towards Hg2+ and melamine

The difficulty of ions and biomolecules detection often lies in the exclusion of the response from

interfering materials. The method with good selectivity is thus of great significance in practical

applications.

Figure 3.16. Selectivity study of SiQDs-Thy towards various metal ions and biomolecules

61

Therefore, several metal ions and biomolecules with good water-solubility in aqueous phase

were selected and their ability to quench the fluorescence intensity of SiQDs-Thy was examined

(Figure 3.16). The FL intensity of SiQDs-Thy was quenched to 51% and 54% of the original

intensity in presence of Hg2+ and melamine respectively. Other metal ions such as Na+, K+, Ca2+,

Mg2+, Fe2+ and biomolecules such as dopamine and glucose could not induce significant

fluorescence quenching although their concentration was kept five times higher than the

concentration of Hg2+ and melamine. These results indicated that SiQDs-Thy can act as a

selective fluorescence sensor for Hg2+ and melamine.

3.11 Conclusion

In recent years, SiQDs have attracted huge research interest due to good biocompatibility, low

cytotoxicity and wide availability of source materials. But the enhancement of selectivity and

sensitivity of SiQDs towards target analyte remains a challenge. On the other hand, Hg2+ and

melamine are very hazardous for human health even at very low concentration. The development

of simple and sensitive detection method for Hg2+ and melamine is of great demand. We

developed novel and highly photoluminescent thymine functionalized SiQDs (SiQDs-Thy) via

EDC/NHS coupling of thymine-1-acetic acid to SiQDs with the aim of sensitive and selective

detection of Hg2+ and melamine. Thymine is ubiquitous in DNA and shows selective interaction

with specific ions and biomolecules. After thymine functionalization, SiQDs-Thy showed

noticeable fluorescence response towards Hg2+ based on T–Hg2+–T metal ion-mediated base

pairs with a low detection limit. Also, melamine immediately quenched the fluorescence of

SiQDs-Thy resulting from hydrogen bond interaction between thymine and melamine. SiQD-

Thy was also highly selective towards Hg2+ and melamine compared to other ions and

biomolecules. In view of these desirable features, SiQDs-Thy are expected to find potential

applications in selectively and efficiently detection of Hg2+ and melamine in real water samples,

food products and biological environments.

62

References

[1] X. Xu, S. Ma, X. Xiao, Y. Hu, and D. Zhao, "The Preparation of High-Quality Water-

Soluble Silicon Quantum Dots and their Application in the Detection of Formaldehyde,"

RSC Adv., vol. 6, pp. 98899-98907, 2016.

[2] Y. Chen, Y. Bai, Z. Han, W. He, and Z. Guo, "Photoluminescence Imaging of Zn(2+) in

Living Systems," Chem. Soc. Rev., vol. 44, pp. 4517-46, 2015.

[3] Y. Li, J. Xu, and C. Sun, "Chemical Sensors and Biosensors for the Detection of

Melamine," RSC Adv., vol. 5, pp. 1125-1147, 2015.

[4] K. V. Chakravarthy, B. A. Davidson, J. D. Helinski, H. Ding, W. C. Law, K. T. Yong, et

al., "Doxorubicin-Conjugated Quantum Dots to Target Alveolar Macrophages and

Inflammation," Nanomedicine, vol. 7, pp. 88-96, 2011.

[5] F. Erogbogbo, C.-A. Tien, C.-W. Chang, K.-T. Yong, W.-C. Law, H. Ding, et al.,

"Bioconjugation of Luminescent Silicon Quantum Dots for Selective Uptake by Cancer

Cells," Bioconjugate Chem., vol. 22, pp. 1081-1088, 2011.

[6] Y. Guo, L. Zhang, F. Cao, L. Mang, X. Lei, S. Cheng, et al., "Hydrothermal Synthesis of

Blue-Emitting Silicon Quantum Dots for Fluorescent Detection of Hypochlorite in Tap

Water," Anal. Methods, vol. 8, pp. 2723-2728, 2016.

[7] X. Zhang, X. Chen, S. Kai, H.-Y. Wang, J. Yang, F.-G. Wu, et al., "Highly Sensitive and

Selective Detection of Dopamine Using One-Pot Synthesized Highly Photoluminescent

Silicon Nanoparticles," Anal. Chem., vol. 87, pp. 3360-3365, 2015.

[8] Y. He, Z.-H. Kang, Q.-S. Li, C. H. A. Tsang, C.-H. Fan, and S.-T. Lee, "Ultrastable,

Highly Fluorescent, and Water-Dispersed Silicon-Based Nanospheres as Cellular

Probes," Angew. Chem., Int. Ed., vol. 48, pp. 128-132, 2009.

[9] A. Shiohara, S. Hanada, S. Prabakar, K. Fujioka, T. H. Lim, K. Yamamoto, et al.,

"Chemical Reactions on Surface Molecules Attached to Silicon Quantum Dots," J. Am.

Chem. Soc., vol. 132, pp. 248-253, 2010.

[10] Y. Li, Z.-Y. Zhang, H.-F. Yang, G. Shao, and F. Gan, "Highly Selective Fluorescent

Carbon Dots Probe for Mercury(II) Based on Thymine–Mercury(II)–Thymine Structure,"

RSC Adv., vol. 8, pp. 3982-3988, 2018.

63

[11] H. Xu, S. Huang, C. Liao, Y. Li, B. Zheng, J. Du, et al., "Highly Selective and Sensitive

Fluorescence Probe Based on Thymine-Modified Carbon Dots for Hg2+ and L-Cysteine

Detection," RSC Adv., vol. 5, pp. 89121-89127, 2015.

[12] J. Du, Z. Wang, X. Peng, and J. Fan, "In Situ Colorimetric Recognition of Melamine

Based on Thymine Derivative-Functionalized Gold Nanoparticle," Ind. Eng. Chem. Res.,

vol. 54, pp. 12011-12016, 2015.